Enzymes As Diagnostic Tools

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Enzymes as Diagnostic Tools

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Enzymes as Diagnostic Tools

Ram Sarup Singh*, Taranjeet Singh*, Ashish Kumar Singh† *Carbohydrate and Protein Biotechnology Laboratory, Department of Biotechnology, Punjabi University, Patiala, India †Biosensor Technology Laboratory, Department of Biotechnology, Punjabi University, Patiala, India

9.1 INTRODUCTION

Enzymes as biocatalysts have been widely used in industrial processes such as food processing, beer fermentation, laundry detergents, pickling purposes, and to control, as well as accelerate, the catalytic reactions in order to quickly and precisely obtain various valuable end products. Moreover, enzymes are widely used both at the laboratory scale, as well as at the commercial level for a wide range of applications, including stereospecific bioconversion, utilization of waste into beneficial end products or environmental friendly substitutes, upgrading raw materials, and so forth. The exact potential of these remarkable catalysts has not yet been fully determined, and thus, the new uses of existing enzymes are being further explored. Enzyme metabolism is a fundamental bio-process that plays a pivotal role in the survival of all species, including humans, plants, animals, and microorganisms, as their specific function is to catalyze chemical reactions. Abnormality in the enzyme metabolic system leads to a number of metabolic disorders. Thus, owing to the remarkable properties of enzymes, they are used for the diagnosis of such disorders. Worldwide, researchers have con- centrated more on clinical applications of the enzymes, such as acid phosphatase, alanine transaminase, aspartate transaminase, creatine kinase, gelatinase-B, lactate dehydrogenase, and so forth. Enzymes act as preferred bio-markers in various disease conditions, such as myocardial infarction, renal disease, liver disease, rheumatoid arthritis, schizophrenia, cancer, and so forth. They provide insight into the diseased condition by diagnosis, prognosis, or by assessment of response therapy. Even though literature reveals the use of enzymes in disease conditions, comprehensive analysis is still lacking. The diagnosis and monitoring of various diseases is very demanding nowadays for routine examination of clinical samples and other associated tests. These require typical analytical methods that demand proficient skill and time for collecting the desired sample volume to

Advances in Enzyme Technology 225 # 2019 Elsevier B.V. All rights reserved. https://doi.org/10.1016/B978-0-444-64114-4.00009-1 226 9. ENZYMES AS DIAGNOSTIC TOOLS perform the clinical tests. The enzymes that are used for the detection/diagnosis or prognosis of disease conditions are called “diagnostic enzymes.” Moreover, due to their substrate specificity and quantitated activity in the presence of other proteins, enzymes are preferred in diagnosis, and therefore can be used as a diagnostic tool for disease detection. A diseased state often leads to tissue damage, depending on the severity of the disease. Under such conditions, enzymes specific to diseased organs are released into blood circulation with enhanced enzyme activity. The measurement of such enzyme activities in blood/plasma, or any other body fluid, has been employed in the diagnosis of diseased tissues/organs. Nowadays, biosensors are becoming popular potential tools for medical diagnostics, path- ogen detection, food safety control, and environmental monitoring. The enzymes are well known as a biological component in the development of biosensors due to their high specificity. Biosensors have become popular because of their accurate, rapid, sensitive, and selective detection strategies, which can be used routinely. In the healthcare sector, liable and correct information on the desired biochemical parameters is very important. In this context, biosensors are providing great solutions to the problems faced by the existing healthcare industry. Biosensors can be applied for rapid detection of different metabolites for the diagnosis of various diseases. This chapter emphasizes the role of enzymes and enzyme-based biosensors as diagnostic tools for various clinical conditions and diseases.

9.2 ENZYMES IN DIAGNOSIS OF DISEASES

The diagnosis of the serum level of certain enzymes has been used as an indicator of cellular damage that results in the release of intracellular components into the blood stream. Hence, when a physician reveals that a person has to undergo a neurological enzyme assay, the purpose is to ascertain whether or not there is brain damage. Commonly assayed enzymes for the diagnosis of various diseases are alkaline phosphatase, creatine kinase, aminotransferases (alanine aminotransferase and aspartate aminotransferase), dehydrogenases (sorbitol dehydrogenase and lactate dehydrogenase), cholinesterase, cyclooxygenase, tartrate- resistant acid phosphatase, and so forth (Table 9.1). Many other enzymes are also involved in human and veterinary medicine for the clinical diagnosis of different diseases. The enzymes that facilitate the rapid diagnosis of various diseases are as follows:

9.2.1 Liver Diseases The liver is the largest internal organ, which plays an important role in many body functions, including detoxification of blood, cholesterol, glucose, iron metabolism, and so forth. Various conditions affect the normal functioning of the liver, including hepatitis, Epstein Barr virus infection, fatty liver, excessive intake of alcohol and drugs such as acetaminophen, and so forth. During these abnormal conditions, various enzymes are released into the bloodstream, and quantification of these catalysts could lead to the detection of disease. Enzymes that help in the diagnosis of abnormal liver/liver damage are described as follows. 9.2 ENZYMES IN DIAGNOSIS OF DISEASES 227

TABLE 9.1 Enzymes in Diagnosis of Diseases

Enzyme Disorder/Disease Reference/s Acid phosphatase Malaria [1]

Alanine aminotransferase Hepatocellular damage [2] Hepatitis B and C [3] Alkaline phosphatase Chronic kidney disease [4] Paget disease or rickets/osteomalasia [5] Type II diabetes [6] Obstructed liver [7]

Amylase Pancreatitis [8] Myocardial infarction [9] Aspartate aminotransferase Hepatic diseases [10] Dental disorder [11] Liver fibrosis [12]

Butyrylcholinesterase Schizophrenia [13] Alzheimer’s disease [14] Parkinson’s disease [15] Cathepsin-D Renal cell carcinoma [16] Dental disorder [17] Breast cancer [18]

Rheumatoid arthritis [19,20] Creatine kinase Myocardial damage [21] Neuroleptic malignant syndrome [22] Cysteine cathepsins Premaligant lesions in colon, thyroid, brain, liver, breast, and [23] prostate Gamma glutamyl transferase Cardiovascular mortality [24] Gelatinase-B Gastric cancer [25] Vascular dementia [26] Rheumatoid arthritis [27]

Malignant gliomas [28] Glycogen phosphorylase-BB Myocardial infarction [29] Glucose-6-phosphatase Gierke disease [30] Hypoglycemia [31]

Continued 228 9. ENZYMES AS DIAGNOSTIC TOOLS

TABLE 9.1 Enzymes in Diagnosis of Diseases—cont’d

Enzyme Disorder/Disease Reference/s Glucose-6-phosphatase Gastric cancer [32] dehydrogenase Lactate dehydrogenase Pyroptosis [33]

Necrosis [34] Breast cancer [35] Leukocyte esterase Periprosthetic joint infection [36] Urinary tract infection [37] Bacterial peritonitis [38] Ascitic fluid infection [39]

Lipase Acute pancreatitis [8,40] Skin disorders [41] Lysozyme Rheumatoid arthritis [7,42] Tuberculous meningitis [43] Tuberculous pericarditis [44] Prostate cancer Prostatic acid phosphatase [45]

Tartrate-resistant acid Osteoarthritis [46] phosphatase Tartrate-resistant acid Giant cell tumor [47] phosphatise-5b Bone metastases [48,49]

9.2.1.1 Aminotransferases Aminotransferases (transaminases) are a group of enzymes that carry the interconversion of amino acids and oxoacids by the transfer of amino groups. Alanine aminotransferase (ALT), formerly known as glutamate pyruvate aminotransferase (GPT), and aspartate aminotransferase (AST), formerly known as glutamate oxaloacetate aminotransferase (GOT), are the two clinically important enzymes classified under aminotransferases.

9.2.1.1.1 ALANINE AMINOTRANSFERASE Among liver disorders, the most prevalent health problems are hepatitis and hepatocirrhosis, which are caused by different factors, including excessive alcohol intake, im- balanced diet, lack of exercise, insufficient sleep, and overconsumption of high calorie food [50]. In clinical diagnosis, the level of ALT (E.C. 2.6.1.2) acts as an important indicator of potential liver disease. Inside cell cytoplasm, ALT catalyzes the reversible transamination of L-alanine and 2-oxoglutarate into pyruvate and glutamate, respectively. In a normal, 9.2 ENZYMES IN DIAGNOSIS OF DISEASES 229 healthy human adult, the ALT concentration range is from 5 to 35 U/L, and its concentration above this range indicates a damaged/diseased liver, heart, and muscle [51]. Earlier, ALT concentration was determined by spectrophotometric, calorimetric, or chromatography methods. Recently, simple and low-cost paper-based analytical devices (PADs) for ALT determination have attracted the significant interest of various researchers [52].

9.2.1.1.2 ASPARTATE AMINOTRANSFERASE The serum level of AST helps people to diagnose damaged body organs, especially the heart and liver. AST (E.C. 2.6.1.1) catalyzes the transamination of L-aspartate and 2-oxoglutarate into oxaloacetate and glutamate, respectively. In a healthy human adult, AST has a concentration of around 5–40 U/L [51]. However, after severe damage, the AST level rises 10–20-times higher than the normal range. AST is also found in the red blood cells, muscle tissue, and other organs, including the kidney and pancreas. It can be used in combination with other enzymes to monitor myocardial, hepatic parenchymal, and muscle diseases in humans and animals. Moreover, to screen the liver fibrosis in chronic hepatitis B, the AST-to-platelet ratio index could be a useful marker, when transient elastography is not available [12].

9.2.1.2 Alkaline Phosphatase (ALP) ALP (E.C. 3.1.3.1) hydrolyzes the phosphate ester bonds in an alkaline environment. The rise in the level of serum ALP is a useful marker of liver disease, particularly cholestatic diseases in which bile ducts are being blocked, as in the case of obstructive jaundice [7].

9.2.1.3 Gamma Glutamyl Transferase (GGT) GGT (E.C. 2.3.2.2) is present in the cell membranes of almost all human cells, and helps in the transport of amino acids from one peptide to another. Therefore, GGT is sometimes also referred to as a transpeptidase. It is found abundantly in the kidney, liver, pancreas, and in- testine, but mainly, GGT is detected in serum that is derived from the liver. Hence, GGT acts as an important biomarker of hepatobiliary disease. Due to toxic or infectious hepatitis, there is a moderate elevation in GGT level (two- to five-fold). In cholestasis, intrahepatic biliary blockage leads to a 5–30 times higher serum GGT than the normal level. Serum GGT can also be elevated in other disease conditions, such as hyperthyroidism, rheumatoid arthritis, myo- tonic dystrophy, and obstructive pulmonary disease. The GGT-to-platelet ratio index pre- sents a novel marker of liver fibrosis in patients with chronic hepatitis [53]. Moreover, research is being conducted to evaluate the use of GGT as a successful biomarker for neph- rotoxicity and cardiovascular disease tests.

9.2.2 Cancer Cancer can be defined as a disease in which group of cells grow abnormally, resulting in their uncontrolled growth and proliferation. This proliferation can be fatal, if it is allowed to continue and spread. Moreover, 90% of cancer related deaths are due to the process called metastasis. Cancer can occur in many different body parts, including the lungs, breast, colon, prostate, brain, mouth, or even in the blood. During the diseased condition, the level of certain 230 9. ENZYMES AS DIAGNOSTIC TOOLS enzymes in the bloodstream becomes abnormal, which further acts as a biomarker of cancer prognosis. A few enzymes that are involved in the detection of cancer are mentioned as follows:

9.2.2.1 Acid Phosphatases (ACP) Five different types of ACP (E.C. 3.1.3.2), namely prostatic, erythrocytic, macrophage, lysosomal, and osteoclastic, are found in humans, and they differ widely with respect to their origin, molecular weight, sequence length, and resistance to tartrate and fluoride level. Acid phosphatase is found throughout the body, but mainly in the prosthetic gland. The prostate gland of the human male has 100 times more ACP level than in any other body tissue. Pros- tatic acid phosphatase is used to monitor the progress of prostate cancer, as it is strongly expressed by prostate cancer cells [45]. Moreover, acid phosphatases are very much concen- trated in semen, thus rape victims are often tested for the presence of acid phosphatase in vaginal fluid.

9.2.2.2 Cathepsin D (CD) CD (E.C. 3.4.23.5) is a ubiquitous lysosomal aspartic protease that breaks down proteins into several polypeptide fragments that digest other lysosomal exo- and endopeptidases. It is synthesized in the rough endoplasmic reticulum as preprocathepsin-D. It is found in nearly all cells, tissues, and organs, but not in lysosome-free RBC. CD is overexpressed by epithelial breast cancer cells, and contributes to the prognosis of breast cancer [54]. It is also involved in other mechanisms, including apoptosis [55], protein degradation [56], processing of hormones, antigen 32, neuropeptides [57], and so forth.

9.2.2.3 Cysteine Cathepsins (CCs) CCs are the lysosomal proteases that are being upregulated in various types of cancers, and are involved in tumorigenic processes such as angiogenesis, apoptosis, and invasion. There are 11 CCs present in the human genome (B, C, F, L, K, V, S, X/Z, H, W, and O), each with distinct expression levels and specificities that contribute to particular physiological responses [58]. CCs such as B, L, and H are distributed ubiquitously, and are stable at neutral pH. Therefore, they are harmful if secreted out of their normal lysosomal localization. The CCs level elevates in tumor conditions, including cancer of the breast, ovary, uterine, cervix, lung, brain, head, and neck. Hence, CCs act as an important marker in cancer and other inflammatory disorders, such as inflammatory myopathies, periodontis, and rheumatoid arthritis [59].

9.2.2.4 Cyclooxygenase-2 (COX-2) COX-2 (E.C. 1.14.99.1) catalyzes the conversion of arachidonic acid into prostaglandins H2, which is the precursor of various molecules, including prostaglandins, thromboxanes, and prostacyclins. Preclinical studies have shown that they are expressed in inflammatory reactions, carcinogenesis, cell proliferation, apoptosis, invasiveness, and immuno-suppression [60]. Moreover, COX-2 is also expressed in response to cytokines, inflammatory mediators, mitogens, and RAS-mediated signaling [61,62]. Recent studies have shown that COX-2 also acts as a biomarker in the progression of tumors, including stomach [63], breast [64], and urinary bladder carcinoma [65]. 9.2 ENZYMES IN DIAGNOSIS OF DISEASES 231

9.2.2.5 Dehydrogenases (DH) DH are the class of enzymes belonging to the oxidoreductases group that oxidizes a substrate by transferring a hydrogen to an acceptor that is either a NAD+/NADP+ or flavin coenzyme, such as FAD/FMN. Hence, they are sometimes called donor dehydrogenases. Two dehydrogenases, namely, sorbitol dehydrogenase and lactate dehydrogenase, are used for cancer prognosis, and are being classified under dehydrogenases.

9.2.2.5.1 SORBITOL DEHYDROGENASE (SDH) Sorbitol dehydrogenase (L-iditol-2-dehydrogenase; E.C. 1.1.1.14) catalyzes the reversible oxidation-reduction between the polyhydric alcohol D-sorbitol and D-fructose using NAD+/NADH as a coenzyme. It is located primarily in the cytoplasm and mitochondria of the human liver, kidney, and seminal vesicles. An abnormal serum concentration of SDH has been reported in prostate cancer [66], and precancerous colorectal neoplasms [67]. Moreover, an enhanced level of SDH can be observed during acute liver damage and parenchymal hepatic diseases.

9.2.2.5.2 LACTATE DEHYDROGENASE (LDH) LDH (E.C. 1.1.1.27) is widely expressed in different human tissues, and it catalyzes the interconversion of pyruvate and lactate during the glycolysis and glyconeogenesis process. LDH gene expression is upregulated in many human malignant tumors, including colorectal cancer [68], lung cancer [69–71], breast cancer [72], oral cancer [73], prostate cancer [74], germ cell cancer [75], and pancreatic cancer [76]. Hence, the prognostic value of the serum LDH level in cancer patients has been considered a significant topic of research recently. Moreover, LDH also acts as a prognostic marker in patients with acute leukemia [77] and sickle cell disease [78].

9.2.2.6 Tartrate-Resistant Acid Phosphatase (TRAP) TRAP (E.C. 3.1.3.2) is a member of purple acid phosphatases containing binuclear iron (Fe2+/Fe3+) that catalyze the hydrolysis of phosphate ester and liberate reactive oxygen [79]. It is highly expressed in osteoclasts, and a lower expression level has been reported in activated macrophages and dendritic cells [80]. TRAP exists in two isoforms, that is, TRAP5a and TRAP5b. TRAP5a is highly expressed in inflammatory macrophages and dendritic cells. An enhanced serum concentration of TRAP5a has been reported in rheumatoid arthritis, kidney disease, systemic lupus erythematosus [81], and breast cancer [82]. TRAP5b is specifically released by bone-resorbing osteoclast cells. Serum TRAP5b activity is also increased in other pathological conditions, such as Paget disease [83], hyperparathyroidism [84], severe osteoporosis [85], multiple myeloma [86], and bone metastasis originated from breast and other cancers [87,88]. Therefore, TRAP5b acts as a potential marker for the diagnosis and prognosis of various types of cancers with high incidence of bone metastasis including breast, prostate, lung, and multiple myeloma.

9.2.2.7 Thymidine Kinase (TK) TK (E.C. 2.7.1.21) is one of the salvage enzymes important for nucleotide metabolism during DNA synthesis. It catalyzes the reversible phosphorolysis of thymidine to thymine 232 9. ENZYMES AS DIAGNOSTIC TOOLS and 2-deoxy-D-ribose. Two isoforms of TK are TK1 and TK2. TK1 is present in cell cytoplasm and is cell-cycle dependent, whereas TK2 is cell-cycle independent and located in cell mitochondria. The TK1 level in human serum is considered a useful marker for screening and diagnosis of various malignancies. An elevated TK1 level has been reported in patients suffering from breast cancer [89], gastric cancer [90], chronic lymphocytic leukemia, acute lymphoblastic leukemia [91], colon cancer [92], uterine cancer [93], and prostate cancer [94]. It can also predict the presence of neoplasia at earlier developmental stages [95].

9.2.3 Cardiac Disorders Cardiovascular diseases are the class of diseases that involve the heart or blood vessels. These are commonly related to atherosclerosis, where fat is deposited in the arteries in the form of plaque, causing them to narrow, and possibly become blocked. This can cause high blood pressure, heart attack, stroke, or peripheral arterial diseases. Various risk factors related to cardiac abnormality include high blood cholesterol, physical inactivity, depression, stress, excess weight, unhealthy eating, and so forth. Apart from these, few enzymes also act as a diagnostic tool for the early detection of cardiac diseases. Their abnormal level in the blood serum acts as an indicator/marker in cardiac disease prognosis.

9.2.3.1 Creatine Kinase (CK) CK (E.C. 2.7.3.2) is an intracellular enzyme that catalyzes the transfer of a phosphate group from creatine phosphate to ADP to generate a molecule of ATP after depletion of ATP in muscle cells. Therefore, extra energy embodied in creatine phosphate is provided to muscles by CK. Similarly, a reversible reaction of creatine phosphate is performed by CK, when muscles are at rest. CK exists in three isoforms, that is, CK-MM, CK-MB, and CK-BB; out of these, CK-MB is the most specific and accurate means of detecting myocardial infarction. In a normal, healthy male, the CK level is 0.038–0.174 U/mL, while in the case of a healthy female, it is between 0.026 and 0.14 U/mL. The serum CK concentration increases to a maximum of up to 2.0 U/mL during myocardial infarction, muscular dystrophy, and inflammatory reactions, thereby helping in the early prognosis of disease conditions. Recently, in addition to myocardial infarction, CK-MB also acts as a biomarker in the diagnosis of uncomplicated hypertension [96] and chronic kidney disease [97]. Nowadays, CK-MB activity assay has been replaced by CK-MB mass assay, in which the protein concentration of CK-MB is measured, rather than its catalytic activity. Researchers around the globe are more interested in immunoassays for measuring CK-MB levels, as these analytical interferences may lead to false positive results.

9.2.3.2 Glycogen Phosphorylase (GP) GP (E.C. 2.4.1.1) is a glycolytic enzyme that plays a pivotal role in carbohydrate metabolism by mobilization of glycogen. GP exists in three isoforms; GPMM, GPLL, and GPBB, with different physiological functions. GPMM is a muscle isoform that supports muscle contraction, GPLL is a liver isoform that maintains blood glucose homeostasis, and finally, GPBB is a brain isoform responsible for the glucose supply during anoxia/hypoglycemia. Among the three isoforms, GPBB is predominant, and acts as a biomarker for anthracycline cardiotoxicity [98], 9.2 ENZYMES IN DIAGNOSIS OF DISEASES 233 acute myocardial infarction [99], coronary syndrome [100], pregnancy, and preterm preeclampsia [101].

9.2.3.3 Gelatinases Gelatinases are the proteolytic enzymes that convert gelatine into polypeptides, peptides, and amino acids, which are further used in many metabolic pathways. Coronary artery diseases followed by myocardial infarction are the major cause of a large number of human deaths in developing countries such as India. Therefore, early detection of such disease conditions is a must. Gopcevic et al. [102] reported the use of gelatinase A (E.C. 3.4.24.24) and gelatinase B (E.C. 3.4.24.35) as a marker for the early phase detection of acute myocardial infarction. Moreover, gelatinase, in association with lipocalin, acts as a potent marker for the diagnosis of acute kidney injury [103], cardiac remodeling after myocardial infarction [104], and myocardial fibrosis [105].

9.2.3.4 Amylases Salivary α-amylase (E.C. 3.2.1.1) is a digestive enzyme that cleaves starch into smaller car- bohydrates by hydrolysis of internal α-1,4-glycosidic bonds. Measuring the α-amylase activity is emerging as a potent biomarker for the detection of heart failure [106], chronic psychosocial stress [107], and monitoring kidney functions [108].

9.2.4 Miscellaneous Enzymes

9.2.4.1 Lysozyme Lysozyme (E.C. 3.2.1.17) plays a pivotal role in the prevention of bacterial infections by attacking peptidoglycan in the bacterial cell wall. Peptidoglycan is composed of the repeating amino sugars, N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM), cross-linked by peptide bridges. Lysozyme hydrolyzes the bonds between NAG and NAM, which increases the bacterial permeability, leading the bacteria to burst. Lysozyme is widely distributed in a variety of tissues, including the liver, articular cartilage, plasma, saliva, tears, and milk. Detection of elevated urinary lysozymes is an indicator of renal damage and nephropathy [109].

9.2.4.2 Butyrylcholinesterase (BChE) BChE (E.C. 3.1.1.8) related to acetylcholinesterase is a serine hydrolase that catalyzes the hydrolysis of esters of choline, including acetylcholine. It is widely distributed in the nervous system, pointing to its possible involvement in neural function. Higher plasma BChE activity has been reported in patients suffering from schizophrenia [13], whereas reduced BChE activity is an early indicator of trauma-induced acute systemic inflammatory response [110]. Moreover, BChE also acts as a potent biomarker of Alzheimer’s disease [14] and cholinesterase depression [111].

9.2.4.3 Lipases Lipases (E.C. 3.1.1.3) are ubiquitous enzymes reported from plants, animals, and microbial sources. Lipases have great potential in medical applications. Studies reported the use of 234 9. ENZYMES AS DIAGNOSTIC TOOLS lipases in substitution therapy, where enzyme deficiency is overcome by their external ad- ministration in diseased conditions. Lipases act as an activator of the tumor necrosis factor, and therefore can be used for the treatment of malignant tumors [112]. Moreover, lipases are also used in the treatment of gastrointestinal disturbances, digestive allergies, dyspepsias, and so forth [113]. Lipases from Candida rugose immobilized on a nylon support was used in the synthesis of lovastatin, a drug that helps in lowering the serum cholesterol level [114].

9.3 ENZYMATIC BIOSENSORS AS DIAGNOSTIC TOOLS

A biosensor is an analytical device that incorporates a biological or biologically derived sensing material with close proximity to the physico-chemical transducer. The main purpose of such a device is to generate a discrete or continuous signal that is proportional to the concentration of the analyte [115]. The first biosensor was developed in 1962 for the detection of glucose, based on immobilized glucose oxidase, using an oxygen electrode as a transducer [116]. Since then, the concept of the enzyme electrode has been popularized and implemented in various other enzyme-based biosensors for the detection or sensing of a particular analyte. Later on, a potentiometric urea biosensor was developed for the detection of urea in clinical samples [117]. The biosensor was constructed by immobilizing urease on the ammonium ion selective electrode as a transducer. Furthermore, a transducing element, such as thermistor (a heat sensing element), has been utilized for the development of a biosensor based on a thermal enzyme probe [118,119]. Lubbers and Opitz [120] coined the term “optode,” which con- sists of a fiber optic sensor with an immobilized indicator to measure carbon dioxide or oxygen. They extended this concept to develop an optical biosensor for alcohol by immobilizing alcohol oxidase on the end of a fiber-optic oxygen sensor [121]. Some of the popular transducer systems used in enzymatic biosensors are tabulated in Table 9.2.

9.3.1 Biosensors: Back to Basics In any biosensor configuration, numerous components are assembled. A generalized schematic representation of a biosensor is shown in Fig. 9.1. The basic principle is to convert a biologically induced recognition event into a usable signal. In order to achieve this, a transducer is used to convert the chemical signal into an electronic one, which can be processed in

TABLE 9.2 Transducer Systems Used in Enzymatic Biosensors

Transducer Example/s Electrochemical Clark electrode; mediated electrode; ion selective electrode (ISE); field effect transistor (FET)-based devices

Optical Photodiodes; waveguide systems; integrated optical devices Calorimetric Thermistor; thermopile Piezoelectric Quartz crystal microbalance (QCM); surface acoustic wave (SAW) devices Magnetic Bead-based devices 9.3 ENZYMATIC BIOSENSORS AS DIAGNOSTIC TOOLS 235

FIG. 9.1 Schematic representation of working of an enzyme-based biosensor. some way, usually with a microprocessor. Over the years, a variety of enzyme-based biosensors have been developed, but only a few of them are commercialized. Most of the published work on enzymatic biosensors focuses on targeted blood glucose monitoring based on amperometric techniques. The amperometric biosensors have been divided into three genera- tions, based on their working principle (Fig. 9.2). The first generation biosensors were projected by Clark and Lyons [116], and implemented by Updike and Hicks [122], who denoted the term “enzyme electrode.” The enzyme electrode described by them was com- prised of an oxidase enzyme, that is, glucose oxidase, immobilized onto a dialysis membrane on a platinum electrode. The depletion of O2, or the formation of H2O2, is subsequently

FIG. 9.2 Historical developments of enzymatic biosensors. 236 9. ENZYMES AS DIAGNOSTIC TOOLS measured by the platinum electrode. The second generation biosensors have been commercialized, mostly in a one-time use testing platform. MediSense (Waltham, Massachusetts, United States) was the first company to launch second generation biosensors as a product. Again, their application was blood glucose monitoring, but this device was only for home use. The mediation was provided by ferrocene species. The third generation biosensors are marked by the progression from the use of a freely diffusing mediator (O2 or artificial) to a system where the enzyme and mediator are co-immobilized at an electrode surface, making the biorecognition component an integral part of the electrode transducer. The co-immobilization of the enzyme and mediator can be accomplished by redox mediator la- beling of the enzyme, followed by enzyme immobilization in a redox polymer, or enzyme and mediator immobilization in a conducting polymer. A plethora of biosensors have been developed to provide diagnostic information on a patient’s health status. The details of different enzymatic biosensors used for clinical diagnosis are listed in Table 9.3.

TABLE 9.3 Enzymatic Biosensors as Diagnostic Tools

Detection Test Disease Enzymes Types of Transducer Analyte Range (mM) Sample Diagnosed Reference/s Glucose Amperometric Glucose 102 to 3.4 Blood Diabetes, [123] oxidase serum, hypoglycemia Up to 12 blood [124] 6.30 to 20.09 plasma, [125] urine, and 2.5 to saliva [126] 32.5 103 and 6.0 to 1.3 102 0.01 to 6.5 [127] 0.01 to 7.0 [128]

Glucose Amperometric 1.0 to [129] oxidase/ 40.0 102 horseradish peroxidase Glucose Amperometric 1.6 to 33.3 [130] dehydrogenase Up to 3 [131] Photoelectrochemical 0.2 to 8.0 [132] Cellobiose Amperometric 0.02 to 30 [133] dehydrogenase Urease Amperometric Urea 1.2 to 352.8 Blood and Renal disease, [134] urine stone in urinary 0.1 to 8.5 [135] tract or even 0.2 to 1.8 bladder tumor, [136] liver malfunction 12.5 102 [137] to 1.0

Continued 9.3 ENZYMATIC BIOSENSORS AS DIAGNOSTIC TOOLS 237

TABLE 9.3 Enzymatic Biosensors as Diagnostic Tools—cont’d

Detection Test Disease Enzymes Types of Transducer Analyte Range (mM) Sample Diagnosed Reference/s 0.5 to 150 [138]

0.1 to 10 [139] 14 to 392 [140] 1 102 [141] to 35 8.4 to 840 [142] 56 to 336 [143] Conductometric 0.003 to 0.75 [144]

Potentiometric 0.5 to 40 [145] 1 103 [146] to 1.0

8 103 [147] to 3.0

Optical 0 to 10 [148] 0.7 to 8 [149] 28 to 1120 [150] Piezoelectric 8 105 to [151] 1.0 5.0 104 to [152] 3.0 Arginine/ Amperometric Arginine 1 103 to Blood Leukemia, [153] urease 1.0 serum cancers 1 102 to [154] 1.0

2.5 102 to [155] 0.35

1.4 to 10 [156] Up to 0.03 [157] Creatininase Amperometric Creatinine 0.2 to 2.0 Urine/ Renal disease, [158]

3 blood thyroid Creatinase/ Amperometric 1.0 10 to serum malfunction and [159] sarcosine 0.8 muscular disease oxidase Up to 4 [160] Up to 2.0 [161]

Continued 238 9. ENZYMES AS DIAGNOSTIC TOOLS

TABLE 9.3 Enzymatic Biosensors as Diagnostic Tools—cont’d

Detection Test Disease Enzymes Types of Transducer Analyte Range (mM) Sample Diagnosed Reference/s 1.0 102 to [162] 0.65

1.0 103 to [163] 1.0 Oxalate Amperometric Oxalate 8.4 103 to Blood Idiopathic [164] oxidase 0.27 serum and urolithiasis and urine various intestinal diseases Asparaginase Optical Asparagine 1.0 107 to Blood Leukemia, throat [165] 1.0 102 serum cancer and acute

7 lymphoblastic 1.0 10 to leukemia [166,167] 1.0 102

1.0 106 to [168] 1 103 0.1 to 10 [169]

Amperometric Up to 10-2 [170] Cholesterol Amperometric Cholesterol Blood Coronary heart [171] oxidase serum disease, 0.6 to 6.3 myocardial and [172] 2.8 to 4.1 cerebral [173] infarction Up to 4.1 (stroke) [174]

Lactate oxidase Amperometric Lactate Up to Blood Hyper [175] 0.1 102 lactatemia, cardiac arrest Up to 0.1 Drug and and [176] biological resuscitation, sample sepsis, reduced 10 to Blood renal excretion, [177] 1.0 104 plasma decreased extra hepatic 2 2.0 10 to Blood metabolism, [178] 2 15 10 intestinal 2.0 103 to Blood infarction and [179] lacticacidosis 1.0 5.0 102 to Blood [180] 0.7 serum Lactate Amperometric 1.0 103 to Blood [181] dehydrogenase 12 102 0.1 to 10.0 Blood [182]

Continued 9.3 ENZYMATIC BIOSENSORS AS DIAGNOSTIC TOOLS 239

TABLE 9.3 Enzymatic Biosensors as Diagnostic Tools—cont’d

Detection Test Disease Enzymes Types of Transducer Analyte Range (mM) Sample Diagnosed Reference/s 0.05 to 10.0 Serum [183]

0.0 to 0.3 Serum [184] 0.1 to 24.8 Serum [185] 2.0 104 to Blood [186] 8 104 1.0 102 to Tumor [187] 5 biomarker Optical 5.0 102 to Cancer cell [188] 10 Uricase Amperometric Uric acid 4.0 103 to Urine Gout, renal [189] 6.4 103 disease, Lesch- Nyhan Up to Serum syndrome, [190] 5.0 104 hypertension 2.5 103 to Serum [191] 8.5 102

0.01 to 0.4 Serum [192] NS Blood [193] 1 103 to Serum [194] 0.4 Ascorbic acid Amperometric Ascorbic acid NS Blood Oxidative stress [195] plasma in smokers Acetyl choline Amperometric Acetylcholine 0.001 to 0.1 Blood Alzheimer’s [196] esterase/ and 0.01 to serum and disease choline oxidase 1.0 brain tissue 12 to [197] 2.7 1011 1.0 104 to [198] 6.0 104 Lipase Amperometric Triglycerides 0.5 to 3.9 Blood Hyperlipidemia, [199] serum, coronary plasma and diseases such as tissue liver obstruction, homogenate diabetes mellitus and endocrine 0.5 to 7.9 [200] Impedimetric 0.2 to 3.3 [201]

NS, not specified. 240 9. ENZYMES AS DIAGNOSTIC TOOLS 9.3.2 Enzyme-Based Biosensors in Clinical Diagnosis

9.3.2.1 Biosensors for Blood Glucose Diabetes is an incurable disease developed from the deficiency of insulin in the body, causing either elevated blood-glucose levels (hyperglycaemia) or low glucose concentration (hypoglycemia). The hormone insulin is synthesized and secreted from the pancreas to mediate metabolic reactions involving glucose. Diabetes has been associated with many medical conditions, including coeliac disease, cystic fibrosis, tuberculosis, and heart disease. Such complications can result in retinopathy leading to blindness, nephropathy giving rise to renal failure, peripheral nerve damage with increasing risks of extremity (foot) ulcers, amputation, cardiovascular diseases, or even cancer. Diabetes has also been described as the “silent epidemic” [202], and since its discovery, great efforts have been made to achieve efficient diagnosis, monitoring, and treatment. Detection of glucose has been the most studied analyte in diabetic patients. The level of glucose can be monitored either in vivo or in vitro. Biosensors for glucose monitoring have been developed using various types of enzymes.

9.3.2.1.1 GLUCOSE DEHYDROGENASE (GDH)-BASED GLUCOSE BIOSENSORS The first glucose biosensor based on glucose dehydrogenase from Erwinia sp. and carbon paste was developed by Laurinavicius et al. [131]. The enzyme was immobilized in a polylysine-albumin gel, and the supporting material was a paste of chemically modified carbon powder, fumed silica, and binding material. The anodic response current of the biosensor on glucose was recorded at 0–200 mV vs Ag/AgCl reference electrode. The linearity of the biosensor can be extended up to 3.0 mM by coating the enzymatic layer of the biosensor with a layer of the polyvinyl alcohol (PVA) emulsion. The developed biosensor showed good linearity at low glucose concentration, due to the elimination of oxygen influ- ence. A new electron-transfer mediator, 5-[2,5-di-(thiophen-2-yl)-1H-pyrrol-1-yl]-1,10- phenanthroline iron(III) chloride (FePhenTPy) oriented to a nicotinamide adenine dinucleotide-dependent-glucose dehydrogenase (NAD-GDH) system was synthesized [130]. Then, with NAD-GDH, a disposable type of amperometric glucose biosensor was constructed with FePhenTPy as an electron-transfer mediator on the screen-printed carbon electrode (SPCE), and its performance was evaluated, where the addition of reduced graphene oxide (RGO) to the mediator showed the enhanced sensor performance. In the real sample experiments, the interference effects by acetaminophen, ascorbic acid, dopamine, uric acid, caffeine, and other monosaccharides (fructose, lactose, mannose, and xylose) were completely avoided by coating the sensor surface with the Nafion film containing lead (IV) acetate. The reliability of the developed glucose sensor was evaluated by the determination of glucose in artificial blood and human whole blood samples. Recently, a disposable and economical biosensor has been developed by electrodeposition of a hybrid quantum dot (ZnS-CdS) onto the surface of a pencil graphite electrode (PGE). GDH was immobilized onto the modified electrode (GDH/ZnS-CdS/PGE) by using glutaraldehyde as a cross-linker. The cyclic voltammetry and photoamperometric measurements have revealed that GDH/ ZnSCdS/PGE is capable of signaling photoelectrocatalytic activity toward NADH, when the surface of GDH/ZnS-CdS/PGE was exposed with a light source. The current produced by the enzymatic reaction in the photoamperometric was found linearly correlated with the glucose concentration. The photoelectrochemical biosensor was successfully applied to the 9.3 ENZYMATIC BIOSENSORS AS DIAGNOSTIC TOOLS 241 real samples. The results showed sensitivity, repeatability, and good selectivity for monitoring glucose with amperometric and photoamperometric transducers [132].

9.3.2.1.2 CELLULOSE DEHYDROGENASE (CDH)-BASED GLUCOSE BIOSENSORS A glucose biosensor based on cellulose dehydrogenase from a mutant of Corynascus thermophilus has been developed, and a glassy carbon electrode (GCE) was obtained by direct electrodeposition of gold nanoparticles (AuNPs). The electrode was further modified by cross-linking to a mixture of self-assembled monolayers of 4-mercaptobenzoic acid (4-MBA) and 4-aminothiophenol (4-APh). The CDH/GA/4-APh,4-MBA/AuNPs/GCE platform showed an extended linear range from 0.02 to 30 mM with sensitivity of 3.1 0.1 μA/mM cm2, excellent stability, and good selectivity for glucose. The modified electrode was successively used as a glucose biosensor exhibiting a detection limit of 6.2 μM. The biosensor was applied for the detection of glucose in human saliva samples, with successful results in terms of both recovery and correlation with glucose blood levels [133]. This further suggests the development of noninvasive glucose monitoring devices.

9.3.2.1.3 GLUCOSE OXIDASE (GOD)-BASED GLUCOSE BIOSENSORS A glucose biosensor has been fabricated by successful entrapment of glucose oxidase into a chitosan matrix, which was cross-linked with glutaraldehyde. The main advantage of this matrix is to provide good biocompatibility and a stabilizing microenvironment around the enzyme. This biosensor shows a fast electrochemical response to glucose. The linear range for glucose determination was 1 105 to 3.4 103 M, with a detection limit of 5 106 M based on S/N ¼ 3. The biosensor also retained 90% catalytic activity after 2 weeks of storage, under dry conditions at 4°C [123]. A novel glucose biosensor has been developed based on the immobilization of GOD by cross-linking in chitosan (CS) on a glassy carbon electrode, which was modified with gold-platinum alloy nanoparticles (Au-PtNPs) by electrodeposition on multiwall carbon nanotubes (MWCNTs) in CS film (MWCNTs/CS). The developed biosensor showed outstanding performance for glucose detection at a low potential (0.1 V) with a high sensitivity (8.53 μA/M), a low detection limit (0.2 μM), a wide linear range (0.001–7.0 mM), a fast response time (<5 s), and good reproducibility, stability, and selectivity. The biosensor was applied for the determination of glucose in human blood and urine samples [128]. Later, another new amperometric glucose biosensor was constructed based on platinum nanoparticles/polymerized ionic liquid-carbon nanotubes (CNTs) nanocomposites (PtNPs/PIL-CNTs). The CNT was functionalized with polymerized ionic liquid (PIL) through direct polymerization of the ionic liquid 1-vinyl-3-ethylimidazolium tetrafluoroborate on the carbon nanotubes, and then used as a support for the highly dis- persed PtNPs. The electrochemical performance of the PtNPs/PIL-CNTs modified glassy carbon (PtNPs/PIL-CNTs/GCE) electrode has been realized by typical electrochemical techniques. The PtNPs/PIL-CNTs/GCE electrode showed high electrocatalytic activity toward the oxidation of hydrogen peroxide. The constructed amperometric glucose biosensor showed good analytical characteristics, such as high sensitivity (28.28 μA/mM/cm2), wide linear range (up to 12 mM), and a low detection limit (10 μM) [124]. An investigation of gold nanoparticles-bacterial cellulose nanofibers (Au-BC) nanocomposite as a platform for the development of an amperometric biosensor for the detection of glucose has been proposed. In this case, two enzymes, that is, glucose oxidase and 242 9. ENZYMES AS DIAGNOSTIC TOOLS horseradish peroxidase, were co-immobilized in an Au-BC nanocomposite-modified glassy carbon electrode. A rapid and sensitive amperometric response to glucose was observed in the presence of an electron mediator. The detection limit for glucose under optimal conditions was as low as 2.3 mM, with a good linear range from 10 to 400 mM. The biosensor has been successfully applied for the determination of glucose in human blood samples [129]. Periasamy et al. [125] proposed the direct electrochemistry of glucose oxidase in a gelatin- multiwalled carbon nanotube (gMWCNT)-modified glassy carbon electrode. GOD was covalently immobilized onto the surface of gMWCNT-modified GCE through a well-known glutaraldehyde chemistry. The immobilized enzyme showed a pair of well-defined reversible redox peaks with a formal potential (E0)of0.40 V, and a peak to peak separation (ΔEp)of 47 mV. The surface coverage concentration (Г) of GOD in gMWCNT/GOD/GA composite film modified GCE was 3.88 109 M/cm2, which indicates high enzyme loading. The electron transfer rate constant (ks) of GOD immobilized onto gMWCNT was 1.08 s1, which validates a rapid electron transfer process. The composite film showed a linear response to 6.30–20.09 mM glucose and a good sensitivity of 2.47 μA/mM/cm2 for glucose. The developed biosensor showed storage stability for 2 weeks. Moreover, it showed negligible response to 0.5 mM of ascorbic acid (AA), uric acid (UA), acetaminophen (AP), pyruvate (PA), and lactate (LA), which shows its potential application in the determination of glucose from human serum samples. The composite film exhibited excellent recovery for glucose in human serum at a physiological pH with good practical applicability. Qiu et al. [126] developed a glucose biosensor by immobilizing GOD by a simple physical adsorption method onto the nanostructured gold thin film fabricated by using electrodeposition and galvanic replacement technology. This provided a facile method to prepare morphology-controlled Au films, and also facilitated the preparation and application of enzyme-modified electrodes. An obvious advantage of the developed biosensor based on the enzyme electrode (GOD/Au/GCE) was that the nano-Au film provided a favorable microenvironment for GOD, and facilitated the electron transfer between the active center of GOD and the electrode. The cyclic voltammetry (CV) results indicated that the immobilized GOD displayed a direct, reversible, and surface-confined redox reaction in the phosphate buffer solution. Furthermore, the enzyme-modified electrode was used as a glucose biosensor, showing a good linear relationship in the concentration range of 2.5–32.5 μM and 60–130 μM with a detection limit of 0.32 μM(S/N ¼ 3) at an applied potential of 0.55 V. Due to the excellent stability, sensitivity, and antiinterference ability, Au thin films are useful in the construction of glucose biosensors. Another biosensor for monitoring glucose in blood has been constructed by immobilizing glucose oxidase onto the surface of an electrochemically reduced graphene oxide multiwalled carbon nanotube hybrid (ERGO- MWCNT) modified glassy carbon electrode. GOD was immobilized onto the ERGO-MWCNT hybrid film; as a result, direct electrochemistry of GOD has been achieved. The results compared with pristine MWCNT have shown a 2.1-fold higher peak current, and very low peak- to-peak separation (DEp) of 26 mV at the hybrid film, which demonstrates faster electron transfer between the GOD and the modified electrode. Moreover, the modified film exhibited high electro-catalytic activity toward glucose via reductive detection of oxygen consumption in the presence of a mediator. The proposed biosensor exhibited a low detection limit of 4.7 μM, with a wide linear range of 0.01–6.5 mM, and acquired good storage and operational stabilities. The accurate glucose determination in human blood serum and better recoveries 9.3 ENZYMATIC BIOSENSORS AS DIAGNOSTIC TOOLS 243 achieved in spiked urine samples have shown great potential for the developed biosensor in practical applications [127].

9.3.2.2 Biosensors for Urea in Blood and Urine During metabolic activity, the urea is synthesized in the liver, and filtered out from the bloodstream by the kidneys every day. Generally, abnormal urea concentration indicates disease related to the liver and kidney, or renal disorder [203]. The detection of urea is important in many areas, including medical, environmental, and food. Rapid and sensitive detection of urea is essential not only in clinical industries, but also in fertilizer industries, as well as in the agriculture sector. It is the predominant final metabolite of nitrogenous compounds in mam- mals, accounting for 80%–90% of nitrogen excretion in humans. So, the monitoring of urea concentration in human blood or urine has become an important diagnostic approach for assessing the condition of the kidneys and associated health effects. Patients suffering from kidney disorder often experience the accumulation of urea in the blood. The typical concentration of urea in normal blood is 2.5–7.5 mM based on the uptake of diet [204]. A significant increase in urea concentration is observed during chronic and acute forms of renal disease (50–70 mM and 120–150 mM, respectively). Such abnormal urea levels can be reduced to 10 mM by hemodialysis or peritoneal dialysis. The urea level in dialysate may vary from 3 to 16 mM [205]. The enzymatic biosensors for urea detection are generally based on urease. The first urea biosensor was developed by Guilbault et al. [117]. They used a cation-selective glass electrode containing urease to obtain a signal for urea based on the formation of ammonium ions by its hydrolysis. Since then, the use of urease as a biocatalyst for the development of urea biosensors has attracted considerable interest, and various types of urea biosensors have now been constructed. Eggenstein et al. [134] developed a potentiometric urea-sensitive biosensor + based on a NH4 sensitive disposable electrode. The developed biosensor showed a detection limit of 20 μM, and a good linear range of 72 μM–21 mM. The biosensor was successfully applied for the direct determination of urea in blood samples. The results obtained from the biosensor showed good agreement with the spectrophotometric method. Castillo et al. [143] prepared homogeneous electroconductive films of PANI-poly(n-butyl methacrylate) composites with a surfactant (PVME or PVEE) by using a casting method. The morphological and mechanical properties of the PANI composite were improved by poly(vinyl methyl ether), PVME, or poly(vinyl ethyl ether), PVEE. The composites with PVME were tested as an ammonia conductometric sensor, and showed good analytical performance. Also, urease was immobilized on this electroconductive film for biosensing of urea, and a linear concentration range was between 0.33 and 2.0 mM. This detection range was adequate for the analysis of urea in serum samples. Kovacs et al. [149] developed a planar wave-guide-type optical urea biosensor based on the detection of ammonium ions liberated during the catalytic reaction with an ion-selective optode membrane that contained nonactin as an ion-selective ionophore, and ETH 5294 chromoionophore in a thin plasticized PVC film. The sensor contained an ammonium- sensitive, 1.0 mm thick optode layer, and a second covering layer made of polyurethane. Ure- ase was immobilized by cross-linking on the surface of the secondary layer. The biosensor achieved a good linear range between 0.08 and 2 mM, as well as a fast response time, which enabled the analysis of about 30 samples per hour. Limbut et al. [138] immobilized urease on 244 9. ENZYMES AS DIAGNOSTIC TOOLS three different SiO2-based matrices, including controlled pore glass (CPG), Poraver, and silica gel. These modified matrices were used in an enzyme column reactor coupled with a conduc- tivity meter for conductometric detection of urea. The three enzyme reactors prepared with the different SiO2-based matrices showed good responses for urea with a linear concentration range between 5 and 45 mM. However, it was observed that the sensitivity of the enzyme reactor column prepared with Poraver was 9.8-times less than for CPG, and 9.2-times less than for silica gel. This may be due to the lower percentage of SiO2 group in Poraver (67%), which was 30% less than CPG (>97%), and 32% less than silica gel (>99%). The SiO2 is important for the linkage between the support and the enzyme. Less SiO2 means less immobilized enzyme, and thus, a smaller response. Also, the surface area might be another factor. Both the particle size and pore size of Poraver were much larger than those of CPG and silica gel. Another urea biosensor based on urease immobilized on DEAE-cellulose paper has been developed. The immobilization technique has increased the half-life of the enzyme over 150 days with no practical leaching over a period of 2 weeks. The developed biosensor showed a good linear range from 10 to 400 mg/dL [150]. Another biosensor was constructed by immobilizing urease onto a piezoelectric alumina membrane based on conductometric response. The developed biosensor showed a wide detection range of 0.5 μM–3.0 mM, bearing a fast response time and reasonable storage stability [152]. A potentiometric array-based biosensor has been used for the determination of urea in the presence of interference ammonium, potassium, so- dium, and hydrogen found in a biological matrix. The sensor is an all solid-state type, employing carboxylated PVC as the polymeric membrane, having a linear range detection of 104 to 102 M urea, with a short response time. This bioelectronic tongue was subjected to the analysis of urine samples, although some discrepancies were observed between real time and test samples [139]. A novel potentiometric urea biosensor has been developed for selective and quantitative recognition of urea by immobilizing urease onto Ti/urease- imprinted TiO2 film, and monitoring the potentiometric response caused by the immobilized urease/urea reaction system. The developed urea biosensor exhibited shorter response time (25 s), wider linear range (0.008–3 mM), lower detection limit (5.01 M), and good stability (with about 93.3% of the original response signal retained after 1 month). The clinical analysis of the urea biosensor confirmed the feasibility of urea detection in urine samples [147].An amperometric biosensor has been developed for the quantitative estimation of urea in aqueous medium using a pH-sensitive dye, hematein. Urease was covalently immobilized onto the surface of gold nanoparticles, with modified electrodes functionalized with hyperbranched polyester-Boltron H40 (H40-Au) coated onto an indium-tin oxide (ITO)- covered glass substrate. The main advantage of this immobilization technique is to provide the resulting enzyme electrode (urease/H40-Au/ITO) with a high level of enzyme immobilization and good stability. The biosensor based on Urs/H40-Au/ITO as the working electrode showed a good linear current response to the urea concentration ranging from 0.01 to 35 mM. The urea biosensor exhibited a sensitivity of 7.48 nA/mM with a response time of 3 s [141]. A miniaturized potentiometric urea lipid film-based biosensor on graphene nanosheets has been constructed. The developed potentiometric urea biosensor exhibited good reproducibility, reusability, selectivity, and rapid response time (4 s), a long shelf-life, and high sensitivity of c. 70 mV/decade over the urea logarithmic concentration range from 1 106 to 1 103 M [146]. An interesting nanomaterial based on multilayered graphene (MLG) has 9.3 ENZYMATIC BIOSENSORS AS DIAGNOSTIC TOOLS 245 been exploited for the development of an electrochemical urea biosensor. The developed biosensor exhibited good linearity of 28–280 mM, sensitivity of 14.34 μA/mM cm2, low detection limit of 11.24 mM, and response time of 10 s [140]. Further, single-enzyme nanoparticles (SENs) with excellent activity and stability were successfully fabricated via the surface modification and in situ aqueous polymerization of a separate urease molecule. A novel piezoelectric biosensor was developed for urea determination based on SENs immobilization onto nanoporous alumina membranes. The developed biosensor showed high selectivity, shorter response time (12 s), good linear range (1.0–80 mM), and the lower limit of detection was 50 mM. The clinical analysis of the urea biosensor confirmed the feasibility of urea determination in urine samples [151]. A urea biosensor based on pH-sensitive field-effect transistors has been developed by immobilization of recombinant urease by entrapment in PVA/SbQ photopolymer. The developed biosensor showed a wide range of linearity 0.5–40 mM and less response time (1–2 min). The real samples of blood serum and hemodialysate without urea caused no biosensor response. The biosensor showed no significant decrease of responses during 5 months of storage [145]. A new urea biosensor has been constructed based on zeolite-adsorbed urease and applied for the analysis of urea in blood serum samples. The linear range of urea determination by using the developed biosensor was 0.003–0.75 mM, and the limit of detection was 3 μM. Good reproducibility of urea estimation in real samples was studied easily (RSD ¼ 10%). Thus, by using this biosensor, healthy people can be distin- guished from people with renal dysfunction [144]. Recently, a copper polyaniline nanocomposite-based transducer as a novel probe for ammonium ions has been constructed and realized for the selective detection of urea. The developed biosensor showed high selectivity toward the urea. The biosensor sensitivity was 112 3.36 mA/M cm2, the biosensors responded linearly over a concentration range 1–125 mM, with a detection limit of 0.5 mM, and a response time of 15 s [137]. An electrochemical urea biosensor has been constructed by electropolymerizing 4-(2,5-Di(thiophen-2-yl)-1H- pyrrol-1-yl)aniline monomer (SNS-aniline) on pencil graphite electrode (PGE), then modifying the polymer-coated electrode surface with di-amino-ferrocene (DAFc) as the mediator, and finally, immobilizing urease by cross-linking. The developed urea biosensor showed a good linear range of 0.1–8.5 mM for the urea, sensitivity of 0.54 μA/mM/cm2, a detection limit of 12 μM, and the response time of 2 s. The developed biosensor was applied with real human blood and urine samples, which showed excellent analytical performance with insignificant interference [135]. A new electrochemical urea biosensor based on ferrocene-poly(amidoamine) (Fc-PAMAM) dendrimers incorporated with multiwalled carbon nanotubes (MWCNTs) has been constructed. The developed urea biosensor exhibited a good linear range of 0.2–1.8 mM, a detection limit of 0.05 mM, fast response of 3 s, and sensitivity of 1.085 μA/cm2/mM. The biosensor was applied for urea detection in real samples from healthy humans, which showed excellent performance in terms of selectivity and sensitivity [136]. A novel amperometric urea biosensor by immobilizing urease onto self-assembled polyamidoamine grafted multiwalled carbon nanotube (MWCNT-PAMAM) dendrimers- modified gold electrode was developed. The biosensor exhibited excellent performance in urea detection at an applied potential of 0.45 V, with the response time of 3 s, a good linear range of 1.0–20 mM, a detection limit of 0.4 mM, and sensitivity of 6.6 nA/mM/cm2. A negligible response was observed in the presence of possible interfering molecules. Nondiluted human blood plasma has also been used for real sample applications, which showed great performance in urea measurement [206]. 246 9. ENZYMES AS DIAGNOSTIC TOOLS

9.3.2.3 Biosensors for Uric Acid Uric acid is produced in the liver cells by the major catabolic pathway of purine degradation. Further, uric acid is not metabolized in the liver, and is excreted by the kidneys and intestinal tract. In human blood plasma, the reference range of uric acid is from 3.6 to 8.3 mg/dL. The plasma level of uric acid is considered an important marker for disorders associated with alterations of plasma uric acid concentration, such as hyperuricemia (gout), leukemia, renal impairment, ketoacidosis, Lesch-Nyhan syndrome, and lactate excess [207,208]. Another study has suggested that uric acid acts as an antioxidant in the human body [209]. So, its measurement for the diagnosis and treatment of some disorders is routinely required. The higher concentration of plasma uric acid has also been reported as a risk factor for cardiovascular disease [210]. The first spectrophotometric method for urate analysis was developed by Offer in 1894. In this method, uric acid was chemically oxidized to allantoin, which reduces the phosphotungstic acid to tungsten blue (a chromophore compound). How- ever, this method have many problems, especially interference due to other species being capable of producing the same reaction [211]. A miniaturized and economical biosensor has been developed for uric acid detection in biological samples. The biosensor was fabricated by using a carbon paste electrode entrapped uricase, and the tetracyanoquinodimethane as redox mediator. The linear range of uric acid determination was 1.0–100 mM, with RSD of 0.20%. The developed biosensor was applied to human blood samples analysis, and it showed good agreement with the results obtained by the colorimetric method. The storage stability of the developed biosensor was achieved up to 4 months (250 assays), with only a 13% decrease in its enzymatic activity [189]. Another novel uric acid biosensor was constructed by electropolymerization of pyrrole and ferrocene on the surface of platinum using the cyclic voltammetry scanning between 0.0 and 1.0 V vs Ag/AgCl, at a scan rate of 50 mV/s. The uricase was immobilized by cross-linking onto the polypyrrole-modified film onto the surface of a platinum electrode. The response time of the developed biosensor against uric acid was 5–6 min on a constant potential of +0.7 V vs Ag/AgCl. The working principal of the biosensor was based on the amperometric detection of H2O2, which is produced in the enzymatic reaction of uric acid. The detection limit of the developed biosensor was achieved up to 5.0 107 M. The biosensor storage remained stable for up to 5 weeks without any significant loss of biosensor response. The detection of uric acid in the biological samples by the developed biosensor has also been achieved [190]. A new electrochemical uric acid biosensor has been developed by immobilizing uricase by cross-linking on polyaniline-polypyrrole (pani-ppy) nanocomposite film-modified platinum electrode. The detection based on the oxidation potential of hydrogen peroxide at 0.4 V vs Ag/AgCl was performed. The linear range of the developed biosensor was found to be 2.5 106 to 8.5 105 M, with a response time of 70 s [191]. A conductometric biosensor for the detection of uric acid has been constructed by immobilizing uricase onto the surface of a platinum electrode. The developed biosensor dem- onstrated a good linear range (1.0–6.0 ppm), and has also shown excellent selectivity. The response time of the developed biosensor was 150 s in aqueous solution and in human blood samples. The developed biosensor was applied successfully to the real sample for the detection of uric acid in blood serum. The storage stability of the biosensor was more than 3 days without loss of significant response [192]. Recently, uricase was immobilized in two different 9.3 ENZYMATIC BIOSENSORS AS DIAGNOSTIC TOOLS 247 polymers; polypyrrole and poly(allylamine). The purpose of this kind of polymer matrix was to construct a selective and more stable biosensor for uric acid analysis. In the biosensor, both the layers were prepared by an electropolymerization technique, using platinum as a working electrode. The enzyme was immobilized with the glutaraldehyde cross-linking chemistry. The performance of the developed system was found to be good, and the biosensor remained stable for more than 30 days without loss of significant response. The results also suggested that a combination of PPy and PAA can be used as the matrix for the biosensor development for uric acid determination in biological samples [193]. A carbon nanotube and chitosan nanocomposite film-based novel amperometric uric acid biosensor has been developed by using a silver nanoparticle-modified gold electrode. The amperometric response for uric acid was found at the potential of 0.35 V vs Ag/AgCl. Under optimal conditions, the fabricated uric acid biosensor has shown a very good linear range 1.0–400 μM, with a detection limit of 1.0 μM(S/N ¼ 3). The storage stability of the developed biosensor was up to 205 measurements, and the storage life achieved was more than 45 days. A low Michaelis-Menten constant of 0.21 mM of immobilized uricase indicates that it had great affinity for the uric acid. When used for analysis of uric acid in serum samples, the results showed good agreement with the data obtained by a standard enzymatic colorimetric method [194].

9.3.2.4 Biosensors for Lactic Acid The importance of lactic acid measurement has increased recently, because of its relation to the specific pathological conditions such as shock, respiratory insufficiencies, and heart disease [212]. Lactic acid is a final product of the glycolysis process in all the tissues [213,214]. Lactic acid in normal humans eliminates very quickly, at a rate of 320 mM/h, mostly by the liver metabolism. The reconversion of lactic acid back to the pyruvic acid then takes place. This action keeps the “basal” level of lactate below 1.0 mM in both arterial and venous blood [215]. So, the rapid and sensitive detection of lactic acid is very important. Biosensors are the device that covers these requirements for the determination of lactic acid in human blood. There are two ways to develop a lactic acid biosensor based on biological materials. One is based on the enzyme lactic acid dehydrogenase (LDH) that catalyzes the conversion of pyruvate to the lactic acid and back, as it is the redox process, so it converts NADH to NAD+ and back, and the transfer of electrons can be measured electrochemically. The other is based on the enzyme lactate oxidase, which catalyzes the oxidation of lactic acid, which forms pyruvate and hydrogen peroxide. Further, the hydrogen peroxide can be determined electrochemically.

9.3.2.4.1 LACTATE BIOSENSORS BASED ON LACTATE DEHYDROGENASE (LDH) Many biosensors based on LDH have been constructed for the sensitive detection of lactic acid in human blood samples. Further, the incorporation of nanomaterials improves the measurement quality in terms of limit of detection and signal-to-noise ratio. Based on the nanostructured (Si4N3) surface, a potentiometric biosensor for the detection of lactic acid has been developed. The biosensor was fabricated by employing an electrolyte membrane insulator semiconductor (EMIS). The surface of the membrane was first modified with the polyacrylic acid (PAA) layer, deposited by plasma enhanced chemical vapor deposition (PECVD), and covalently linked to dNH2 groups of the enzyme LDH. Second, the nanostructures were formed onto the modified surface by the colloidal lithography method. The electrochemical 248 9. ENZYMES AS DIAGNOSTIC TOOLS performance of the developed biosensor, in terms of sensitivity, was found to be 49.7 mV/ decade. The linear range was upto 105 M with a detection limit of 2 107 M for the lactic acid in aqueous media [175]. Later, the application of multiwalled carbon nanotubes (MWCNT) was evaluated as a transducer, stabilizer, and for the immobilization matrix or the development of an electrochemical biosensor based on LDH and a redox mediator. The electrochemical response was based on the electrocatalytical properties of the redox mediator to oxidize NADH, which was generated in the enzymatic reaction of lactate with NAD+, under catalysis of LDH. The developed biosensor exhibited a wide linear range from 0.10–10 mM. The developed biosensor was used for direct determination of lactate in the blood samples [182]. Similarly, many other lactate biosensors were developed based on different electrode materials, such as screen printed electrodes [181,183], glassy carbon electrodes [184] and NanoCeO2/GCE [186]. Recently, an optical biosensor based on Nile- Blue-functionalized quantum dots has been constructed. When it was self-assembled in to a hydrogel, it specifically detected and imaged the extra cellular lactate metabolism [188].

9.3.2.4.2 LACTATE BIOSENSORS BASED ON LACTATE OXIDASE (LOD) Lactate oxidase is a flavoenzyme that is useful for the enzymatic determination of lactic acid. Lactate oxidase catalyzes the oxidation of lactic acid using molecular oxygen to produce pyruvate and hydrogen peroxide as the end products. Lactate oxidase contributes to endog- enous H2O2 levels in the blood plasma. Lactate oxidase has long lasting applications in the development of biosensors. A novel lactate biosensor based on ZnO nanoparticles (nanoZnO) was decorated on multiwalled carbon nanotubes (MWCNTs), and the prepared nano-hybrids and nanoZnO-MWCNTs were immobilized on the surface of a glassy carbon electrode to fab- ricate nanoZnO-MWCNTs modified glassy carbon electrodes. The developed lactate biosensor was used for determination of lactate in human blood plasma samples [177]. Similarly, a novel lactate biosensor was developed by fabrication of a gold-coated glass substrate, a growth of ZnO nanorods, and the potentiometric response of lactic acid. The biosensor was fabricated by cross-linking lactate oxidase on the ZnO nanorods. The developed biosensor has shown a good linear range of concentration from 1.0 104 to 1.0 mM, with accept- able sensitivity of about 41.33 1.58 mV/decade. In addition, the developed biosensor exhibited a rapid response time <10 s [176]. Later on, a gold electrode modified with diamond nanoparticles (DNPs) was used for the fabrication of an electrochemical biosensor for lactic acid. The linear range from 0.05 to 0.7 mM, and a sensitivity of 4.0 mA/mM with a detection limit of 15 μM were obtained [180].

9.3.2.5 Biosensors for Arginine L-Arginine is involved in various stages of the cell division process, which significantly in- fluences the leukemic condition. Some animal and human tumors require arginine for their growth (auxotrophic tumors), while normal cells are able to synthesize arginine from citrulline using enzyme argininosuccinate synthase (ASS). Melanoma and hepatocellular carcinoma (HCC) do not express ASS, and therefore are unable to synthesize arginine [216]. Tumor cells require more arginine to generate nitric oxide, which helps to promote tumor angiogenesis [217]. Generally in leukemic patients, the arginine level is low, as compared with a normal one. The plasma arginine level in different types of cancer varies, such as breast cancer at 80 3 μM, from the normal level of 103 9 μM; colon cancer at 80 3 μM, from the 9.3 ENZYMATIC BIOSENSORS AS DIAGNOSTIC TOOLS 249 normal level at 96 7 μM; and pancreatic cancer at 76 5 μM, from the normal level at 99 7 μM [218]. The plasma arginine level lowers in esophageal cancer to 41.9 13.4 μM from the normal level, 83.8 26.8 μM [219]. This can be used as a marker for the diagnosis of esophageal cancer. An ammonium ion-selective electrode is enzymatically sensitized for the detection of creatine and L-arginine. Enzyme layers are formed for both urease and arginase/creatinase, depending on the analyte (substrate), that is, creatine or arginine. The linear range of both the biosensors was 1.0 104 to 3.0 102 M, and the detection limit was below 105 M [153]. Membrane and diffusion potential has also been studied using the Donnan effect. The bienzymatic approach for the detection of arginine was used for both the enzymes arginase and urease. Both the enzymes were co-immobilized onto the ion-selective, electrode- 5 3 based transducer. L-Arginine was measured in the range of 1.0 10 to 1.0 10 M, with a sensitivity of 50 mV/decade [154]. In another study, arginase and urease were immobilized into a gelatin membrane by cross-linking. The developed biosensor showed a good linear re- 5 4 sponse on an L-arginine concentration ranging from 2.5 10 to 3.1 10 M, with a response time of 10 min [155]. Recently, based on the use of Ni(OH)2 nanoparticle-modified carbon nanotubes, an electrochemical sensor for the detection of L-arginine, L-ornithine, and L-citrulline in urine and serum samples has been developed [157]. The main drawback of this method was that it can be used for detection of a group of amino acids, but was not found to be selective for L-arginine.

9.3.2.6 Biosensors for Asparagine L-Asparaginase is widely used as a drug to control tumor progression in acute lymphoblastic leukemia and lymphosarcoma. The growth of normal cells is independent of the require- ment of L-asparaginase. The amino acid asparagine is an essential amino acid for the growth of tumor cells, because these cells lack the asparagine synthetase that synthesizes L-asparagine. In the presence of L-asparaginase, the tumor cells are deprived of an important growth factor, and cannot survive further. The major proposed application of L-asparaginase as an injectable drug, and elimination of L-asparagine in blood, involves the monitoring of L-asparagine in the blood of treated cancer patients (especially ALL patients) to avoid reoccurrence. Biosensor technology can be an alternative promising technology to detect L-asparagine at nanolevels in blood samples. A thermostable recombinant L-asparaginase isolated from Archaeoglobus fulgidus was cloned and expressed in Escherichia coli as a fusion protein. The enzyme was purified and immobilized onto the surface of an ammonium selective electrode (ISE) to develop a biosensor for L-asparaginase [170]. An optical biosensor for the detection of asparagine was developed by co-immobilization of a phenol red indicator with asparaginase. The detection limit of asparagine achieved by using different immobilization matrices, such as a nitrocellulose membrane, was 1010 M, and with silicon gel, it was 1010 M, and with calcium alginate beads, it was 109 M. Furthermore, the developed system has been applied for the detection of asparagine in normal and leukemia blood samples [165–168]. Recently, a ratiometric fluorescent L-asparagine (Asn) biosensor was developed using an oxazine 170 perchlorate (O17) ethyl cellulose (EC) membrane, and the enzyme was entrapped into its matrix. The principle of the developed biosensor was based on the hydrolysis reaction of asparagine under the catalysis of asparaginase, which produces ammonia. The produced ammonia reacts 250 9. ENZYMES AS DIAGNOSTIC TOOLS with the O17-EC membrane, which changes the fluorescence intensities at λem 625 nm. The linear range of developed biosensors for asparagine was 0.1–10 mM, and the detection limit was 0.074 0.0023 mM. The sensing membrane also exhibited good quality in terms of response time, reversibility, and the storage stability. The developed method was successfully applied for the detection of asparagine in the human blood samples [169].

9.3.2.7 Biosensors for Creatinine In our body, the creatinine (2-amino-1-methyl-5H-imidazol-4-one) is the end catabolism product. The extent of the creatinine level in human blood and urine is clinically important, because it partially reflects the renal, muscular, and thyroid functions. The measurement of the creatinine level in blood is also useful for the biomedical diagnosis of acute myocardial infarction, and the quantitative description of hemodialysis therapy. In contrast to the urea level, the concentration of creatinine in biological fluids is not influenced by the protein metabolism, and thus is a more reliable indicator for renal function. The normal creatinine level in human blood ranges from 40 to 150 μM, but it can exceed 1000 μM in certain clinical conditions. The creatinine level in blood >500 μM indicates severe renal disorder, ultimately leading to dialysis or transplantation. Levels <40 μM indicate a decrease in muscle mass. So the measurement of creatinine levels in blood is significant for the diagnosis of these kinds of diseases. A miniaturized enzymatic biosensor for determination of creatine and creatinine in serum was developed [161]. The creatine biosensor is based on the bienzyme sequence reaction, involving creatine amidinohydrolase (CI) and sarcosine oxidase (SO). In the case of the creatinine biosensor, a third enzyme, creatinine amidohydrolase (CA), was used. The current response was generated in both the biosensors based on the oxidation of the hydrogen peroxide formed in the enzymatic layer. The immobilization of these enzymes is generally achieved by the layer by layer deposition using cross-linking with glutaraldehyde. Another, highly sensitive electrochemical creatinine biosensor based on the adsorption of three enzymes onto the polypyrrole modified platinum electrode has been developed. In short, the multienzyme system (creatininase, creatinase, and sarcosine oxidase) is adsorbed on the platinum black matrix. After drying, a thin gelatin layer is cast on the enzyme-adsorbed film and cross-linked under dry conditions by dipping into a diluted solution of glutaraldehyde for 30 s. The linear range was achieved up to 5 mM of creatinine level, with the detection limit of 1–2pM [160]. Many other tri-enzyme-based electrochemical creatinine biosensors have been developed using different nanomaterials-based electrodes, such as a carbon paste electrode [158], a thin film of lead oxide [163], a new zinc oxide nanoparticles/chitosan/carboxylated multiwall carbonnanotube/polyaniline (ZnONPs/CHIT/c-MWCNT/PANI) composite film synthesized on platinum [162], and iron oxide nanoparticles/chitosan graft-polyaniline (Fe3O4-NPs/CHIT-g-PANI) composite film electrodeposited on the surface of a platinum electrode [159]. A comparative analysis of developed creatinine biosensors is listed in Table 9.3.

9.3.2.8 Biosensors for Oxalic Acid Oxalic acid is a metabolite of TCA cycle in the cells. The determination of oxalate in urine and blood is of great interest, as it is required in the diagnosis and medical management of idiopathic urolithiasis and various other intestinal diseases [220]. Many analytical methods 9.3 ENZYMATIC BIOSENSORS AS DIAGNOSTIC TOOLS 251 have been reported for the determination of oxalate, which are bulky, time consuming, less sensitive, and require pretreatment of the sample [221]. Therefore, the demand for a simple, sensitive, accurate, and rapid method has been raised. A highly sensitive, specific, and rapid amperometric oxalate biosensor was developed by covalently immobilizing sorghum leaf oxalate oxidase onto the surface of a platinum electrode modified with carboxylated multiwalled carbon nanotubes, and the conducting polymer, polyaniline nanocomposite film using N-ethyl-N-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxy succinimide (NHS) chemistry. The developed oxalate biosensor exhibited a good linear response range of 8.4–272 mM for oxalic acid, with a correlation coefficient of 0.93, and a rapid response time of 5 s at the applied potential of 0.4 V vs Ag/AgCl. The sensitivity was approximately 0.0113 A/M, with a detection limit of 3.0 mM. The amperometric oxalate biosensor has been successfully used for the analysis of human urine samples [164].

9.3.2.9 Biosensors for Cholesterol Cholesterol is an essential lipid biomolecule found in every cell membrane of all animal and human cells [222]. It is also considered one of the main components of the body system, and a precursor for other biomolecules, such as steroids and hormones. The second-highest death-causing cardiac disease is atherosclerosis (thickening of arteries due to the deposition of cholesterol in the inner walls of arteries), which is caused by several factors. Thus, the estimation of the cholesterol level in blood is essential for the valuation of atherosclerosis and other lipid-related diseases, and for the estimation of the risk of myocardial infarction and thrombosis [223]. The normal concentration of total cholesterol in the blood of a healthy person should be <5.17 mM, and this value can vary from person to person, depending upon age, weight, and gender. The high level for total cholesterol concentration in the human blood is >6.21 mM [224]. Thus, a highly sensitive cholesterol estimation to differentiate these levels is very important for medical diagnostics of related diseases. Most of the developed biosensors use cholesterol oxidase (ChOx), which specifically catalyzes the oxidation of cholesterol by molecular oxygen, and produces 4-cholesten-3-one and hydrogen peroxide, and the produced hydrogen peroxide is determined electrochemically. An amperometric cholesterol biosensor based on the direct electron transfer from the cholesterol oxidase was developed by covalently immobilizing the enzyme functionalized onto the multiwall carbon nanotubes (MWNTs)-modified screen printed electrode (SPE). The bioelectrode (MWNTs/ChOx/SPE) has been characterized using an electrochemical method, including cyclic voltammetry (CV) and chronoamperometry (CA). The incorporation of carbon nanotubes has improved the direct electron transfer of ChOx. The developed biosensor has shown good electrochemical behavior and operational stability. In addition, due to the use of a low potential 0.4 V for the electron transfer from enzyme to electrode, it causes no interferences effect of common compounds in real samples, such as ascorbic acid (AA), uric acid (UA), 4-acetamidophenol (AP), and so forth [172]. Later, Pundir et al. [173] developed an epoxy resin membrane with immobilized cholesterol oxidase mounted on the cleaned platinum electrode, with a parafilm to construct a cholesterol biosensor. The developed biosensor showed an optimal response time of 25 s. The linear operational range of the cholesterol biosensor was 1.0–8.0 mM. The detection limit of the constructed biosensor was achieved up to 1.0 mM. The biosensor showed great storage stability of 6 months at 4°C. This biosensor had an advantage over the existing biosensors, as it works at a comparatively lower 252 9. ENZYMES AS DIAGNOSTIC TOOLS potential. The use of an epoxy resin membrane as a support for immobilization of cholesterol oxidase has resulted in an improved amperometric cholesterol biosensor. A reagentless cholesterol detection in human plasma with a new single-enzyme, membrane-free, self-powered biosensor, in which both cathodic and anodic bioelectrocatalytic reactions are powered by the same substrate, has been developed. The enzyme cholesterol oxidase was immobilized by the entrapment method in a sol-gel matrix onto both the cathode and anode electrode. The generation of hydrogen peroxide, by the enzymatic reaction of cholesterol, was electrocata- lytically reduced by the use of a dye (Prussian blue) at the cathode. Simultaneously, cholesterol oxidation was catalyzed by mediated cholesterol oxidase at the anode. The combination of these two electrodes resulted in a self-powered biosensor with improved sensitivity (26.0 mA/M/cm2), compared with either of the two individual electrodes, and a dynamic linear range up to 4.1 mM for the cholesterol [174]. A new approach was used for the development of a cholesterol biosensor based on two-dimensional gold nanostructure (AuNs) as working electrode material. The AuNs have been synthesized by using the β-diphenylalanine (β-FF) as a sacrificial template, and gold nanoparticles (AuNPs) were synthesized by the ul- trasound irradiation method. The enzyme cholesterol oxidase (ChOx) was immobilized onto the surface of AuNs, which was further attached onto the graphite (Gr) electrode by chem- isorption onto the thiol-functionalized graphene oxide (GO-SH). The final enzyme electrode Gr/GO-SH/AuNs/ChOx has been characterized using different electrochemical techniques, such as cyclic voltammetry, electrochemical impedance spectroscopy (EIS), and chronoamperometry (CA). CV results indicate a direct electron transfer between the enzyme and the surface of modified electrodes. The proposed biosensor exhibits quick response time, high sensitivity, good linear range, and a low detection limit. The proposed biosensor has also been applied successfully to detect cholesterol in the serum samples [171].

9.3.2.10 Biosensors for Triglycerides A natural lipid molecule in our body, known as triacylglycerols, or triglycerides (TGs), is made up of one glycerol molecule that is joined to three molecules of fatty acids (saturated/ unsaturated or both) through ester bonds. The main role of TGs is the significant transporta- tion of dietary fats throughout the blood circulation system. The normal level of TGs in the blood of a healthy person is <150 mg/dL, while 150–199 mg/dL level is considered border- line high, and 200–499 mg/dL is considered a high level of TGs. The elevated TGs level >500 mg/dL in the blood serum is used as a biomarker for cardiovascular disease [225], Alzheimer’s disease [226], pancreatitis [227], and diabetes, due to abnormal lipoprotein metabolism. Considering the clinical importance of its determination in blood serum, a number of biosensors have been developed. Most of them are enzyme-based biosensors, and the enzymes are lipase, glycerol kinase, and glycerol-3-phosphate oxidase. Minakshi and Pundir [228] developed a triple-enzyme-based amperometric biosensor by mounting a cellulose acetate membrane on a platinum electrode. A linear response was obtained for a triolein concentration ranging from 0.2 to 3.5 mM. Pundir et al. [229] developed an electrochemical biosensor by using a mixture of enzymes (glycerol kinase and glycerol phosphate oxidase) co-immobilized with horseradish peroxidase onto the polyvinyl alcohol membrane by glutaraldehyde cross-linking chemistry. The multienzymatic biosensor exhibited a good linear relationship with the concentration of TGs in the range of 0.56–2.25 mM. Later, another amperometric TG biosensor was constructed based on covalent 9.3 ENZYMATIC BIOSENSORS AS DIAGNOSTIC TOOLS 253 co-immobilization of lipase, glycerol kinase, and glycerol-3-phosphate oxidase onto chitosan and zinc oxide nanoparticles composite film [230]. The developed biosensor realized the determination of TGs in human blood samples. Recently, Panky et al. [231] developed a single- enzyme-based biosensor for the detection of tributyrin, and the enzyme lipase was immobilized onto the surface of cerium oxide thin film that was coated on the transparent conducting oxide electrode. This biosensor showed good linearity with a tributyrin concentration in the range of 0.33–0.98 mM. In another investigation, Wu et al. [232] modified a glassy carbon electrode with lipase nanoporous gold biocomposite nanomaterials. The linear response of the developed system was obtained for tributyrin concentrations ranging from 50 to 250 mg/dL. A novel nanomaterial, polyaniline nanotubes (PANI-NT)-based film was electrochemically deposited onto the surface of indiumtinoxide (ITO) coated glass plate for covalent immobilization of lipase by crosslinking to detect TGs using an electrical impedance technique. The detection principle is based on the fatty acid molecules generated during TG hydrolysis, which resulted in the change of charge transfer resistance (RCT) of PANI-NT film with varying TG concentrations. The developed biosensor exhibited a good linear range from 25 to 300 mg/dL, and sensitivity of 2.59 103 K/Ω/mg/dL, with a response time of 20 s [201]. Recently, another lipase-based TGs biosensor was developed. To construct this biosensor, graphite rods were modified with activated carbon (AC) and used as support for the immobilization of lipase. Then, chitosan was used to develop a film onto the bioelectrode, and also to retain the immobilized enzyme. To improve the linear range of TGs detection, the activated carbon was functionalized with carboxyl, and then amine groups (AAC) to enhance the iso- electric point of AC. The linear response was in the range of 50–350 mg/dL, with a detection limit of 9.9 mg/dL. The biosensor showed excellent sensitivity of 0.16 μA/mg/dL [199].

9.3.2.11 Biosensors for Acetyl Choline Acetylcholine (ACh) is an ester compound that is synthesized from choline (Ch) and acetyl coenzyme A (acetyl CoA) by the choline acetyl transferase (ChAT). The chemical name of ACh is 2-acetoxy-N,N,N-trimethylethanaminium. ACh offers transmission the messages between brain nerve cells [233]. The role of Ach is related to many neurological disorders, such as dementia, Alzheimer’s disease, Parkinson’s disease, and schizophrenia [234]. ACh also af- fects parasympathetic nerves and causes the heart rate to slow, growing saliva, and an increase in bladder movements. Thus, due to these reasons, the determination of the level of ACh is very important in clinical studies. ACh also has a significant role in the enhancement of sensory perceptions on waking [235], and in sustaining attention [236]. Damage to the cholinergic (acetylcholine-producing) system in the brain has credible association with the mem- ory deficits associated with Alzheimer’s disease [237]. ACh also promotes REM sleep [238]. There are many enzymatic biosensors in which dual enzyme reactions occur [239]. ACh measurement is completed by oxidation of H2O2, which occurs after AChE and ChO reactions [240]. A bienzyme amperometric biosensor for acetylcholine and choline is based on liquid chromatography (LC) as a detector. The biosensor was fabricated by co-immobilization of acetylcholine esterase and choline oxidase by cross-linking onto the surface of a platinum working electrode attached with a thin-layer electrochemical flow cell. The linear response of the developed biosensor was observed over at least four decades, and absolute detection limits 254 9. ENZYMES AS DIAGNOSTIC TOOLS

(at a signal-to-noise ratio of 3) were 12 and 27 fM injected for the Ch and ACh, respectively. The working stability of the system was 1 month. The possible application of the developed approach was established by the simultaneous determination of Ch and ACh in tissue homogenates of rat brains [197]. Another biosensor was constructed by co-immobilizing acetylcholinesterase (AChE) and choline oxidase to two different electrodes, that is, nanomaterial multiwalled carbon nanotube (MWNT)-modified glass carbon electrodes (GCE) and carbon-screen printed electrodes (SPE). The analytical range of the biosensor based on a GCE and SPE was 1.0–10 μM and 10–100 μM, respectively. Acetylcholine in human blood samples was detected by using the developed biosensor [196]. Recently, a novel electrochemical ACh biosensor was developed by co-immobilizing of acetylcholinesterase and choline oxidase (AChE-ChO) on electrochemically polymerized polyaniline- polyvinylsulfonate (PANI-PVS) film by cross-linking. The ACh detection was based on the electrochemical oxidation of enzymatically produced H2O2 at +0.4 V vs Ag/AgCl. The linear range of the developed biosensor was 1.0 107 to 6.0 107 M [198].

9.4 ALLOSTERIC ENZYME-BASED BIOSENSORS

Allosteric enzymes exhibit regulatable catalytic activities upon the binding of an effector molecule to a receptor site of the enzyme that is different from the active site. In some cases, modulation occurs through binding to distinct, alternative sites, either inhibitory or stimulatory [241]. Allosteric enzymes that catalyze the formation of easily detectable products are potential biosensors (Fig. 9.3). The receptor site acts as the recognition element, the active site as the transducer element, and the whole enzyme integrates both parts through its own struc- ture, and transmits the binding signal via conformational changes. Natural allosteric enzymes, however, cannot be directly used as biosensors, because most of their modulators are devoid of analytical interest. However, it can be possible to incorporate it by modular engineering. Allosteric biosensor prototypes that have been constructed are the result of a trial- and-error approach, rather than of rational design. Some allosteric enzyme-based biosensors are listed in Table 9.4.

FIG. 9.3 Working principle of an allosteric enzyme-based biosensor. 9.4 ALLOSTERIC ENZYME-BASED BIOSENSORS 255

TABLE 9.4 Allosteric Enzyme Biosensors as Diagnostic Tools

Types of Enzyme Transducer Substrate Effector Disease Diagnosed Reference/s Alkaline Piezoelectric BCIP Antibody Swine flu [242] phosphatase β-Galactosidase Optical X-gal and ONPG Antibodies HIV, hepatitis C [243–245] β-Lactamase Optical CCF4-FA Antibodies and HIV, hepatitis C [246,247] streptavidin, ferritin and L-galactosidase Neural Optical Peptidic substrate Short ssDNA Viral diseases [248] protease with fluorophore and a quencher

Ribozymes Optical RNA Short ssRNA Viral diseases [249]

BCIP, 5-bromo-4-chloro-3-indolyl phosphate; CCF4-FA, cephalosporin core linking B7-hydroxycoumarin to fluorescein; ONPG, ortho- nitrophenyl L-D-galactopyranoside; X-gal, 5-bromo-4-chloro-3-indoxyl-L-D-galactopyranoside.

9.4.1 Allosteric Biosensors Based on Engineered Enzymes

9.4.1.1 β-Galactosidase β-Galactosidase catalyzes the hydrolysis of lactose to glucose and galactose, but many lactose analogs producing colored, fluorescent, or luminescent compounds are also formed. The availability of such alternative compounds, especially those that are chromogenic, such as 5-bromo-4-chloro-3-indoxyl-L-D-galactopyranoside (X-gal) or ortho-nitrophenyl L-D-galactopyranoside (ONPG), have strongly supported the use of this enzyme as a reporter for the detection of gene expression, as a marker for the construction of recombinant, plasmid design, and as a partner in fusion proteins for structural stabilization, process monitoring, and purification. Among the series of hybrid enzymes in which an antigenic peptide of foot and mouth disease virus VP1 capsid protein was displayed in the solvent exposed site, two of them exhibited relevant modulation upon binding of the antipeptide monoclonal antibodies [243]. Remarkably, in these two allosteric constructs, the viral peptide has been inserted closely to the active site, and like in the other insertional mutants, their specific activity was lower than the wild-type enzyme. A related series of the enzyme L-galactosidase biosensors, displayed an antigenic peptide from the human immunodefi- ciency virus (HIV-1) gp41 structural protein, also rendered an excellent response against the human serum, when it was compared with the commercial diagnostic immunoassay method [244,245].

9.4.1.2 Alkaline Phosphatase Alkaline phosphatase from E. coli is a homodimer, nonspecific phosphomonoesterase whose activity is highly appreciated for analytical purposes, because of its colorimetric detection. The insertion of an HIV peptide from the structural protein gp120 in the vicinity of the active site rendered a fully active enzyme, but the presence of antipeptide antibodies inhibit 256 9. ENZYMES AS DIAGNOSTIC TOOLS the catalytic rate up to 40%–50% [246]. Recently, a wireless magnetoelastic (ME) biosensor immobilized with E2 glycoprotein was developed to detect classical swine fever virus (CSFV) E2 antibody. The detection principle involves a sandwich complex of CSFV E2-rabbit anti-CSFV E2 antibody-alkaline phosphatase conjugated goat antirabbit IgG formed on the ME sensor surface, with biocatalytic precipitation used to amplify the mass change of antigen-antibody specific binding reaction, induces a significant change in resonance frequency of the biosensor. Due to its magnetostrictive feature, the resonance vibrations and resonance frequency can be actuated and wirelessly monitored through magnetic fields. The experimental results showed that resonance frequency shift increases with the augmentation of the CSFV E2 antibody concentration. Scanning electron microscopy, energy-dispersive spectroscopy and fluorescence microscopy analysis proved that the modification and detection process are successful. The biosensor showed a linear response to the logarithm of CSFV E2 antibody concentration ranging from 5 ng/mL to 10 μg/mL, with a detection limit of 2.466 ng/mL, and sensitivity of 56.2 Hz/μg/mL. The study provides a low-cost, yet highly-sensitive and wireless method for selective detection of the CSFV E2 antibody [242].

9.4.1.3 L-Lactamase The introduction of two independent point mutations in the enzyme inverted the sensing response by promoting enzyme activation upon antibody binding (Table 9.4). The performance of both up and downresponsive biosensors was further confirmed by using an antigenic peptide from the hepatitis C virus; the resulting constructs responded consistently with those carrying the HIV epitope [246]. A bacteriophage-transported TEM-1 L-lactamase was submitted to random insertional mutagenesis to explore permissive sites for foreign peptide display, and the resulting libraries were screened by bio-spanning on immobilized monoclonal antibodies against the prostate-specific antigen [247].

9.4.1.4 Neural Protease A rare case of rational strategy in the development of enzymatic biosensors has been en- couraged in the principle of intrasteric regulation that regulates some natural enzymes. For example, in a partially synthetic construct containing the enzyme cereus neural protease, a phosphoramidite inhibitor covalently binds through a short and flexible single-stranded (ss) DNA hinge, and leads to blocking the activity of the enzyme. The presence of a comple- mentary DNA molecule hybridizing with the hinge DNA segment, and restricting its flexi- bility, promotes the release of the inhibitor and switches on the activity of the cereus neural protease. The results of this activity are dependent on the concentration of DNA molecules with a specific sequence, and can be detected by fluorescence through the hydrolysis of peptide substrates containing both a fluorophore and a quencher [248].

9.4.1.5 Ribozymes The nucleic acids exhibit variations in its catalytic activity upon binding to the different ligands, especially in the small organic effector molecules. This allosteric property of the enzyme ribozyme was further improved by protein engineering, and realized for the development of biosensors and biochips [249]. However, an exclusively modular rational method seems to insufficient for excellent performance of the biosensor. 9.5 COMMERCIALLY-AVAILABLE ENZYME-BASED BIOSENSORS 257 9.5 COMMERCIALLY-AVAILABLE ENZYME-BASED BIOSENSORS

In the past few decades, efforts have been made by academia and industry to develop enzyme-based commercial biosensors for diagnostics. Presently, more than 500 companies worldwide are working in the field of biosensors and bioelectronics [250]. The major commercial sector for practical applications of biosensors is captured by the Yellow Springs Instru- ment Company, Ohio, United States. The first commercial biosensor (glucose biosensor) was launched in 1975. It was based on the electrochemical detection of hydrogen peroxide, and the enzyme used for the development of the biosensor was glucose oxidase. Later, Clem- ens et al. [251] introduced a new amperometric glucose biosensor in a bedside artificial pancreas, and it was marked by Miles (Elkhart, Indiana) under the brand name “Biostator.” Although it is no longer commercially available, a few semicontinuous catheter-based blood glucose analyzers have recently been introduced by VIA Medical San Diego, California. In the same year, La Roche (Basel, Switzerland) launched a lactate analyzer LA 640, in which a redox mediator hexacyanoferrate was used to transfer electrons from lactate dehydrogenase to the electrode. Liedberg et al. [252] proposed an immunosensor based on an immobilized antibody on the surface of a piezoelectric or potentiometric transducer, and later, it was commercialized. In 1987, other types of electrode materials, such as screen-printed enzyme electrodes, were launched by MediSense (Cambridge, Massachusetts), with a pen-sized meter for home blood glucose monitoring. Some of the commercially available enzymatic biosensors for clinical diagnostics are listed in Table 9.5. Currently, Bayer (Fernwald, Germany), Bochringer Mannheim (now Roche Diagnostics, Basel, Switzerland), and LifeScan (Milpitas, California, United States) are now competing for the enzyme-based diagnostic biosensors market, and the combined sales of these companies is dominating the world market. They are rapidly replacing the traditional technologies of diagnostics with biosensors.

TABLE 9.5 Commercial Enzymatic Biosensors for Clinical Applications

Enzyme Linear Range used Analyte (mM) Biosensor Name Company Reference Cholesterol Cholesterol NS Cholesterol Lifestream http://www. oxidase monitor Technologies Inc., lifestreamtech.com United States Glucose Glucose NS Gluco Watch Cygnus, Inc., https://www. oxidase United States cygnusaero.com 1–45 23A/23L YSI; Yellow https://www.ysi. Springs com Instruments, United States

1–40 Autostat GA-1120 Daiichi, Kyoto, https://www. Japan daiichisankyo.com 0.6–60 ADM 300/ECA Eppendorf, FRG, https://www. 20 ESAT 6660 Germany eppendorf.com NS One touch ultra LifeScan, France http://www. lifescan.com

Continued 258 9. ENZYMES AS DIAGNOSTIC TOOLS

TABLE 9.5 Commercial Enzymatic Biosensors for Clinical Applications—cont’d

Enzyme Linear Range used Analyte (mM) Biosensor Name Company Reference NS Accu-check Roche Diagnostics, https://www. Germany roche-diagnostics. co.in

0.05–1.6 103 Glucometer Elite Bayer Diagnostics, https://www. XL Germany bayer.in NS TetraSensFreeStyle Abbott, United https://www. States abbott.com NS Medisens Abbott, United https://www. States abbott.com NS I-STAT Abbott, United https://www. States abbott.com 0.5–50 ZWG Zentrum fur http://www. Wissenschaftlichen wissensgeschichte- Geratebau, Berlin, berlin.de GDR Up to 27 Gluco-20 Fuji Electric, https://www. Tokyo, Japan fujielectric.com NS Glucometer Pulsatum Health https://www. Care Ltd., India pulsatom.com NS Glucometer EKF Industrie- https://www.ekf. Elktronik GmbH, de Germany

Lactate Lactate 0–15 23A/23L YSI; Yellow https://www.ysi. oxidase Springs com Instruments, United States 0.2–30 103 NS Pinnacle https://www. Technology Inc, pinnaclet.com United States

NS Biosens040/ EKF Industrie- https://www.ekf. Lactate Scout+ Elktronik GmbH, de Germany 0.5–12 LA640 LaRoche, Basel, https://www. Switzerland roche.ch/en.htm 1–30 ADM 300/ECA Eppendorf, FRG, https://www. 20 ESAT 6660 Germany eppendorf.com 1.2–18.7 Lacate analyser Accusport1, http://lactate.com United States 0.8–23 Lactate Pro KDK lactate http://www. LT-1710 analyzer, Japan arkray.co.jp/ english/products

Continued 9.7 CONCLUSIONS AND PERSPECTIVES 259

TABLE 9.5 Commercial Enzymatic Biosensors for Clinical Applications—cont’d

Enzyme Linear Range used Analyte (mM) Biosensor Name Company Reference 0.3–25/0.5–25 Lactate plus/ Lactate Analyser, http://www. Lactate Scout United States lactate.com

Glucose, NS Bioscanner 2000 DEX Blood https://www. cholesterol, Glucose Meter, dexcom.com HDL, blood United Kingdom ketone, triglycerides, hemoglobin Uricase Uric acid 0.1–1.2 ADM 300/ECA Eppendorf, FRG, https://www. 20 ESAT 6660 Germany eppendorf.com

9.6 ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) IN DIAGNOSTICS

ELISA is also known as a solid-phase enzyme immunoassay that is used to detect the presence of a specific protein (antigen or antibody) in blood samples. The basic principle of ELISA is to use an enzyme to detect the binding of antigen (Ag) or antibody (Ab). Enzymes such as alkaline phosphatase, horseradish peroxidase, lactoperoxidase, and β-galactosidase are used in ELISA. Three basic principles involved in ELISA are: (i) the antigen-antibody reaction, in which the presence of Ag or Ab is detected in a sample; (ii) the enzymatic chemical reaction, in which a rate of formation of Ag-Ab complex is used to determine the quantity of either Ag or Ab involved in the reaction. Here, the enzyme catalyzes the colorless substrate to produce a colored product; and (iii) signal detection and quantification, in which the intensity of the colored product generated by the enzyme and substrate is detected and measured. ELISA may be used to diagnose various diseases such as HIV, Lyme disease, pernicious anemia, tuberculosis, Rocky Mountain spotted fever, rotavirus, squamous cell carcinoma, syphilis, toxo- plasmosis, chicken pox, zika virus, and so forth. The enzyme-multiplied immunoassay test (EMIT) is used in disease diagnostics. In this assay, the activity of malate dehydrogenase is assayed for the detection of thyroxine by enzyme-linked immunoassay.

9.7 CONCLUSIONS AND PERSPECTIVES

Enzyme and enzyme-based biosensors are valuable tools for the clinical diagnosis of various diseases due to their high specificity, sensitivity, rapid response, ease of self-testing, portability, and so forth. The high specificity of enzymes advocates their choice for medical diagnostics. A literature survey reveals plenty of reports on the use of enzymes in clinical diagnostics. Most of the research conducted on enzymes in diagnostics is carried out at the laboratory scale. So, it is most important to commercialize the applications of enzymes used in diagnostics of various diseases. A number of enzyme-based biosensors have also been 260 9. ENZYMES AS DIAGNOSTIC TOOLS constructed for the diagnosis of diseases. The most successful commercialized enzymatic biosensor is the glucose biosensor, which is used for the detection of glucose levels in blood samples. With the advances in enzyme technology, there is no doubt that enzymes and enzyme-based biosensors will become a powerful tool for the detection of other diseases such as cancer, heart failure, epilepsy, and so forth in the near future. Most enzymatic biosensors work well at the laboratory scale, however, they present many complications during the analysis of real samples. So, it is necessary to develop novel surface modification strategies onto the enzyme electrode in order to eliminate the nonspecific adsorption at surfaces of the enzyme electrode. Moreover, for the diagnosis of large number of samples, multiplexing is required, which can save the assay time. Therefore, it is essential to develop the microarray, as well as the electrochemical devices that can simultaneously perform the analysis of a large number of samples. The sample size is also of utmost importance in clinical diagnosis. There- fore, it is necessary to construct miniaturized enzymatic biosensors using thermostable enzymes that will increase its portability, and can also meet the need of point-of-care (POC), lab-on-chip (LOC), and field application. To fulfill this goal, recombinant enzymes and lab-on-chip techniques offer a solution. So, enzyme and enzyme-based biosensors can be applied for the detection of very low-level targets within an appropriate time.

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