Nanomaterials for Intracellular Ph Sensing and Imaging

Nanomaterials for intracellular pH sensing and imaging

Ying Lian, Wei Zhang, Longjiang Ding, Xiao-ai Zhang, Yinglu Zhang, Xu-dong Wang* Department of Chemistry, Fudan University, 200433, Shanghai, CHINA Western Chemistry bld. 114, Handan Road No. 220, Shanghai [email protected]

Abstract: Intracellular pH is a vital parameter that precisely controls cell functionalities, activities and cellular events. Abnormal intracellular pH is always closely related to the healthy status of cells, which is further translated into pathological changes in a macro perspective. Because of the highly compartmentalized structure inside cells, the pH in each compartment can be precisely tuned to optimize certain cellular functionality, and biological reactions in these regions occur at optimum condition. Thus, it is important to design sensors that can precisely measure pH in these regions, and sensors must have good biocompatibility, physical stability, high sensitivity, wide measurement range, as well as fast response, to fulfill requirements for intracellular pH measurement. In this chapter, we will start from illustrating the importance of measuring intracellular pH, and further discuss how to design optical nanosensors for sensing and imaging intracellular pH. The state of the art technology in intracellular pH sensing and imaging will be reviewed, nanomaterials that are used for constructing intracellular pH sensors will be summarized and the perspective of nanomaterials for intracellular pH sensing and imaging will be given at the end.

1. Overview of the history of pH measurement pH is the abbreviation for Latin "Pondus hydrogenii", Pondus stands for power and hydrogenii stands for hydrogen. In chemistry, the pH scale is a numeric index used to specify acidity and alkalinity of aqueous solutions. The pH value is calculated from the negative log of hydrogen ion concentration in aqueous solution, which is a very important measure in vast application areas, including but not limited to chemistry, human biology, oceanography, steel industry, agriculture and so on. The history of pH measurement can be traced back to the fact that people began to realize that different food have distinct taste. In early time, people believe that food tasted sour belonged to the category of acids and the bitter taste food was classified as bases. Others similar to sodium chloride were classified as a kind of salt. It has been a long time that people using their own sense of taste to distinguish acids and bases. Especially for farmers who directly taste land soil and judge the acidic or basic conditions, cause many diseases. Thanks to Robert Boyle, the famous British physicist and chemist, who accidentally discovered a very useful indicator, litmus, to distinguish acids and bases in 1648. Boyle observed that the color of gillyflowers changed into red when exposed in hydrochloride acid, and returned into its initial blue color in basic solutions. Based on his observation, Boyle invented the very first pH test strip, which has been widely used in the next four centuries. Even today, this brilliant invention is still in common use in modern laboratories. The measurement is very simple and low cost, a drop of test solution was dropped onto the pH test strip, a distinct color change was observed and the pH value can be read via comparison with the standard color card. This outstanding invention ends up the long history of judging acid and bases using our own taste buds, and opens the gate for monitoring acidity and alkalinity using artificial tools. Since its first invention, pH test stripe becomes the most widely-used analytical tool in all kinds of applications. However, the accuracy of the method is not very high and pH test stripe should not be wetted by water before used. The modern definition of pH value was initiated by the Danish chemist Søren Peder Lauritz Sørensen, who was the first person to relate pH values to the concentration of hydrogen ions, and introduced the concept of pH in 1909.1 He then revised the concept into modern pH in 1924 to accommodate definitions and measurements in terms of electrochemical cells. pH, as a simple expression of the acidity and alkalinity of solution, is a function of hydrogen ion concentration and water dissociation. [ ]+[ ]− 퐻 푂퐻 −14 = 퐾퐻2푂 = 1 × 10 [퐻2푂] pH is calculated as the negative logarithm value of hydrogen ion concentration. pH = − lg [퐻]+ Usually, pH value ranges from 0 to 14. However, the pH value of a solution can also be less than 0, which indicates that the concentration of hydrogen ions in solution is already greater than 1 mol/L. It is more convenient to use the concentration of hydrogen ions directly. Pure water can decompose into the same amount of hydrogen ions and hydroxyl ions.

+ − H2O → [퐻] +[푂퐻] The concentration of these ions is equivalent to 10-14 mol/L. [퐻]+[푂퐻]− = 10−14 In neutral solutions, the concentration of hydrogen ions is 10-7 and the pH value of the solutions is equivalent to 7. Solutions with a pH less than 7 are acidic and solutions with a pH greater than 7 are basic. Pure water is neutral, at pH 7 (at 298K), being neither an acid nor a base. With the discovery of acid-base indicators, pH value can be simply measured in the way of titration with the aid of these pH indicators. The end point of titration is indicated by color change of the acid-base indicator. This approach is still widely in use in chemistry practical course in university laboratories, but is not commonly used in other applications, which simply because the method is hardly leading to the invention of instrumentation. Modern laboratories and test facilities are equipped with much simple, compact and even hand-held devices, called pH meter or conductance meters. The very first commercial pH meter was designed by Arnold Beckman in around 1936. His pH meter composes all components of modern devices, including a glass electrode, a reference cell and an electrometer.2,3 The pH meter builds up a small voltage difference between the ion-selective electrode and the reference electrode, and pH value can be deduced from the voltage reading according to the calibration curve.4 Because its compact size, simplicity of use, and low-cost of fabrication, pH meter is still the most widely used and reliable device for measuring pH values. However, this device still has several shortcomings, including the need of constant calibration, since it cannot give reproducible electromagnetic field over longer periods of measuring. Its fragile structure and slow response leads to long measurement time, and poor reproducibility.5-8 The measurement requires large sample volume and the size of glass electrode is simply too big to measure pH inside cells.9,10 Although needle-type pH electrode has been invented for intracellular pH measuring,11 its size is still large compared with the size of a cell, and the inserting the electrode into a single cell always requires specific technique, and at the same time, causes physical damage to cell membrane. The introduction of nuclear magnetic resonance (NMR) provides a non-invasive and non-destructive approach for imaging intracellular pH. This approach relies on the measurement of concentration ratio of protonated and deprotonated forms of phosphate groups, which is a function of pH and allows for simple calculation of intracellular pH. Intracellular molecules, such as ATP and ADP etc, can act as intrinsic reporters, and no other reagent is needed for pH measuring. The first measurement of pH using 31P NMR was conducted by Moon and Richards in red blood cells in 1973.12 Since then, 31P NMR becomes a method of choice in measuring pH value in cytoplasm and cell fluid according to chemical shifts. However, the instrumentation for NMR is rather bulky, complex and expensive. In addition, the technique suffers from its low sensitivity, and high concentration of cells or long data acquisition time is required. In contrast to the aforementioned sensors, optical pH sensors, especially fluorescence-based sensors, have received wide attention during the last decades because of good flexibility, compact size (in nanometer range), requirement of small sensing volume, tunable sensitivity and measurement range.13,14 More importantly, the method is capable to measure the distribution of pH inside cells, which can achieve a high throughput imaging for statistical analysis. 2. Methods for pH measurements Since plenty of chemical and biological processes are highly depending on pH values, lots of effort has been devoted to develop different pH-sensing techniques. Traditional methods for measuring pH values can be roughly grouped into three categories: pH test strips, acid-base titration (used along with color-changing indicator) and electrochemical methods (pH electrodes). Some new techniques represented by optical pH sensors have become more attractive and broaden the scope of pH detection. Though pH test strips are straightforward and simple, the defect of lacking accuracy has limited their applications. In comparison, other methods, including titration, pH electrode and optical pH sensor, are more accurate. In this section, we will briefly discuss the working principle, merit and disadvantages of these pH-sensing approaches. 2.1 Titration Titration is a widely used analytical technique to determine many species, such as acids, bases, oxidants, reductants and metal ions. The acid-base titration can be traced back in late 18th century France.15 A complete acid-base titration, like all other titrations, usually consists of an analyte at unknown concentration, a titrant (or standard solution, a reagent of known concentration) and an indicator (Figure 1).16 In a typical acid-base titration, a certain amount of analyte (or titrant) is placed in the Erlenmeyer flask with pH indicators. The titrant (or analyte) filled in the burette is added dropwisely with stirring. An end point is reached when the color of pH indicator changes. The concentration of analyte is calculated from the consumed amount of titrant. However, the color change at the end point and the amount of consumed titrant is read by human eyes. Artificial errors can hardly be avoided, especially when the color change is not very distinct. Though this method is classic and effective, the requirement of indicators and professional operation, the lack of accuracy and incapability of instrumentation have limited its applications only in chemistry practical courses. The method is hardly being used for analyzing pH values in situ and inside cells. It may be useful for measuring pH of cellular extract or products after cell lysis. For example, by using improved acid-base titration method, the surface acid/base amount on the surface of living marine alga cells could be accurately measured.17

Figure 1. Simple description of a traditional acid-base titration process.16 Reproduced from Ref. 16 with permission from The Royal Society of Chemistry. 2.2 pH electrode

Nowadays, pH values are often measured via pH electrodes, these including quinhydrone electrode and glass electrode. The electrochemical reactions involving hydrogen or hydroxide ions cause voltage difference compared with reference electrode, and the concentration of hydrogen ion can be calculated according to Nernst equation.18 Cyclic voltammetry is a powerful tool for studying the electrochemical redox process, in which the potential applied to working electrode is swept linearly between the anodic and cathodic peak potentials. For pH-dependent redox couple, changing the solution pH would alter the concentration of one of the redox couple, resulting in a shift in redox potential and presenting in the cyclic voltammogram (Figure 2).19

The quinhydrone electrode is a typical representation of pH electrodes based on redox reactions. The electrode consists of an inert metal electrode (usually a platinum wire) in contact with quinhydrone crystals and aqueous solution. Quinhydrone is a charge-transfer adduct, dissolving to form a mixture of quinone and hydroxyquinone with equal concentration. Both of them can easily be transformed to each other during redox reactions, establishing an electrochemical equilibrium as showed in Figure 3.20 It is often used with a pH-independent reference electrode (e.g., silver chloride electrode). The potential difference between the two electrodes is measured and the pH value is calculated according to the calibration curve. However, the quinhydrone electrode is not reliable in basic solution (pH above 8), as well as in solutions with strong oxidizing or reducing reagents, proteins, or high concentrations of salts. Because of the high complexity of intracellular composition, pH sensor based on quinhydrone electrode tends to exhibit poor selectivity and unreliable for intracellular measurement.

Figure 3. The electrochemical equilibrium of quinhydrone occurred at an inert electron conductor 20. Copyright © 2005, American Chemical Society. The pH-sensitive glass electrode is the most widely used pH meter due to its excellent performances, including ideal Nernstian response, immune to interferences, short balancing time, low detection limit, inert to redox system, long-term stability and high reproducibility. The heart of a pH glass electrode is one of the ion-selective electrode made of a doped glass membrane that is sensitive to hydrogen ions. The resistance of the pH-sensitive glass membrane is 108 Ω,21 which varies considerably over temperature, making it only useful at limited temperature ranges (from -25 to 130 °C).22 Other drawbacks, including the mechanical fragility, pressure dependence and signal drift during long-term storage, also limit its applications. The large sample volume requirement and the big size of glass electrodes have made them incapable of intracellular pH measuring. Some pH microelectrodes and ultramicroelectrodes are explored, and their sizes are under 1 μm, which are capable to measure pH inside cells.23 However, their invasive nature, complicated fabrication process, difficulty in precisely insert operation, and relatively large size limited their intracellular application. 2.3 Optical methods Compared with pH electrodes, optical methods for measuring pH possess many advantage, such as immune to electrical interference, feasibility of miniaturization (down to nano-dimension), remote sensing, and capability of high throughput intracellular sensing and imaging. However, like other methods, optical pH sensors also have their own limitations, such as they only respond over a limited range of pH and suffer from cross sensitivity to ionic strength. A typical optical pH sensor consists of a pH indicator with distinct absorbance or fluorescence property changes at different pH. These indicators may be directly used for measuring intracellular pH. However, most of them cannot directly enter intracellular compartment, and have to be modified with certain trans-membrane motif to mediate their transfer into cells. Alternately, these indicators can be immobilized on certain carriers and produce optical sensors for pH. The immobilization of pH indicators is the key step and determines the characteristics of optical pH sensors.24 Three methods, including adsorption, covalent binding and entrapment, are widely used for immobilizing these pH indicators. The physical absorption method is very simple but not reliable, because adsorbed indicator suffers from leakage. The covalent binding method is reliable because pH indicators are covalently bound on a solid substrate. Normally, pH indicators are immobilized on the surface, and are exposed directly in the solution in order to facilitate their response to pH. In most cases, a reference dye is immobilized along with the pH indicator to eliminate interferences during signal generating and reading process (such as light source intensity variation, optical alignment and so on). The signal generated by immobilized indicator can be read out via spectroscope. As for color-changing indicators, absorption spectroscope is employed for signal analysis. This method needs very simple instrumentation, but requires high concentration of pH indicators or a thick sensing layer. Therefore, its sensitivity is not satisfy, even though it is simple and easy to use. In contrast, fluorescence-based pH sensors are much more sensitive, and very low amount of indicators are required. The instrumentation for fluorescent pH sensor contains a light-source to excite the pH sensor and the reference dye. The generated luminescence passes through optical filters and is recorded by photo diode, which is further converted into electric signal. The pH values, as well as the distribution of pH in a large area can be calculated and imaged according to the calibration curve. Optical sensors can be developed into different formats. In case of intracellular pH sensing, optical sensors are always being fabricated into the form of fiber optic or nanosized sensors. These sensors require very small sample volume, and have fast response because of their compact size. Kopelman and co-workers developed the first submicron fiber-optic pH sensors in 1992, enabling the pH measurement in a very small volume of sample with response time as short as milliseconds.25 Imaging fibers comprised of thousands of individual fibers have also been developed and applied on pH measuring with a high spatial resolution.26 Microsized pH-sensitive particles are synthesized that can be spread on a remote sample substrate, and used to determine local pH values with fluorescence signal detected by a GRINscope.27 Nanosphere sensors named PEBBLEs were developed, and injected into individual biological cell for intracellular pH measurement.28 More importantly, optical chemical sensors possess the unique property of multiple sensing at a specific site. Optical chemical sensors for multiple sensing contains pH values have been recently investigated.29 Examples include dual sensors for pH and oxygen30-35 or pH and temperature,36-40 a planar triple sensor for simultaneously measuring pH, oxygen and temperature, and a quadruple sensor layer for oxygen, pH, carbon dioxide and temperature.35 Therefore, applying these optical pH sensors for intracellular sensing is becoming the most attractive challenge. 2.4 Other methods In addition to the above-mentioned pH measurement approach, it is worth mentioning that 31P NMR spectroscopy has emerged as an attractive tool for intracellular pH determination. The chemical shift of inorganic phosphate in the cytosol was accurately correlated with pH in the range between 6.0 and 7.3, and thus could be used as a dynamic 31P NMR probe in biological pH studies.41 On the whole, 31P NMR spectroscopy is a quantitative and fast method for biological analysis without prior separation. It features as non-invasive, but has problems of low sensitivity, requiring high cell densities.42 The continuous innovation and development of pH measurement has been witnessed in the last century since it is widely performed in chemical and biochemical lab. The exploration of new materials and technologies has broadened the development and application of pH sensors, especially for intracellular pH measurement. 3. The importance of intracellular pH sensing Hydrogen ions inside cells play a vital role in the life span of organisms. Many physiological processes and biochemical reactions are highly accompanied by changes in pH. Most of life events require maintenance of acid-base homeostasis in cells, such as cell proliferation and apoptosis, ion transport, endocytosis, and muscle contraction.43,44 Thus, accurate measurement of intracellular pH value is of great significance for studying cell physiology and pathology. 3.1 pH variation in different region Generally, the extracellular pH is 7.4, and the intracellular pH is 7.2, with only a slight decrease. The intracellular pH is a little different from theoretical value of 6.4 calculated according to the Nernst equation, taking into account the existence of transmembrane voltage.45 This is because that intracellular pH is in a state of dynamic equilibrium, maintained by a series of rapid response mechanisms like the active and passive transmembrane transport of ions.46 The intracellular microenvironment is highly compartmentalized. Cells have evolved compartmentalization to provide distinct microenvironment conditions for optimal operation of individual metabolic pathways. As physiological function varies between different organelles and different regions in cell, the corresponding pH values differ a lot, in the range of 4.5 - 8.0. The most acidic part is lysosome, where pH value is 4.5 - 5.5.43,47 As a membrane-bound vesicle, lysosome contains a large amount of various hydrolytic enzymes. Special proteins on the membrane allow hydrogen ions to accumulate inside lysosome. An optimal reaction condition is provided by the acidic lumen for digestion and degradation of almost all kinds of biomolecules. In addition, such an acidic environment is also a necessary for lysosome to realize biological processes, including plasma membrane repair, cell homeostasis, energy metabolism and the immune response.48 Similarly to lysosome, endosome also has an acidic environment, with a pH around 6.5. The low pH is an indicator that organelle is often associated with physiological functions like degradation of biomolecules.43 As for endosome, it mainly involves in proteins sorting, transport and degradation. In contrast, cells also developed alkaline compartment during evolution. Mitochondria, known as the ‘powerhouse’ of a cell, is a double membrane-bound organelle and is the primary site for aerobic respiration which provide the major energy for cellular metabolic activities. The pH inside mitochondria is around 8.0, which is closely related to the role of mitochondria in the cell. During the process of producing adenosine triphosphate (ATP), the efflux of proton is driven by pH potential across the inner membrane due to the proton gradient, which keeps the normal process and maintains the alkali condition inside mitochondria.47,49 Except for energy conversion, mitochondria also contributes to the storage of calcium ions, apoptosis and other biological processes.47 3.2 pH related cellular functionality Intracellular pH differs from organelles, and is strongly related to specific cell function. Intracellular pH regulation is an essential part in adjusting cell functionality and is dependent on biological process. As mentioned above, the actual intracellular pH is higher than theoretical prediction. There is a driving force that causes H+ influx from outside the membrane and OH- efflux. This process is called passive transport. Passive transport is driven by ion gradient across membrane, without the need of energy, usually causing chronic intracellular acid loading. This passive inflow of H+ has little effect on intracellular pH reduction because the concentration of hydrogen ion is quite low. However, long-term accumulation leads to intracellular acidification (Figure 4).46 In order to counteract the intracellular acid loading, acid extrusion is induced by active transport processes. In contrast to passive transport, active transport consumes energy to transport ions against gradient. There are five main regulators to reduce intracellular

45 + - - + acidity by active transmembrane transport: (l) a Na /HCO3 - Cl /H exchanger, (2) a

+ + - - + + Na /H exchanger, (3) a Cl /HCO3 exchanger, (4) the K /H exchange pump of the stomach, and (5) the electrogenic H+ pump of tight epithelia. The influx and efflux of H+ and other related ions together maintain the pH balance and intracellular pH stability. A B

Figure 4. A. The passive efflux of conjugate base anion leads to acid load in the cell.

+ - - + 46 B. Four models for acid extrusion of Na /HCO3 -Cl /H regulator. © Springer-Verlag 1983.

Most cells have the tendency to intracellular acidification. In addition to the acid load described above, cell metabolism can also induce acid accumulation. Taking ATP production as an example, aerobic respiration produces ATP while generating CO2. The amount of H+ consumed when ATP is produced is equivalent to that released by ATP hydrolysis, which results in the presence of CO2, producing net hydrogen ions. In the absence of oxygen, the reaction itself does not alter the intracellular pH, but the resulting ATP increases intracellular acidity in the subsequent hydrolysis.50 Therefore, in this ATP-based universal energy conversion process, the intracellular pH falls. We have known that there is a large difference in intracellular pH distribution in space, and in time. The intracellular pH is constantly changing within cell cycle. Cell proliferation increases when intracellular alkalization, and cell activity decreases when the intracellular acidity increases. Al-Rubeai proposed an early event of intracellular pH reduction in cell apoptosis and demonstrated that intracellular pH changes contributed to the monitoring of cell physiological status and apoptosis.51 In turn, intracellular acidification can also induce apoptosis.52 The interaction between intracellular pH and apoptosis may be associated with inhibition of Na+/H+ exchangers.53 The intracellular pH is also a regulator of nutrient transportation. The endocytosis process is strongly influenced by local temperature and pH.54 Sakai suggested that intracellular pH increases facilitated dynamin-dependent endocytosis and intracellular acidification inhibits endocytosis.55 According to Chin's research, intracellular pH plays an important role in the muscle fatigue mechanism.56 Ca2+ sensitivity of the contractile proteins reduces probably due to acidification, thus making force falls more rapidly. Lagadic-Gossmann pointed out that under the condition of diabetes, intracellular pH influenced contractile force to a less extent based on the relationship between the control of Ca2+ and H+.57 In addition, changes in pH are prevalent in cellular activities such as signal transduction, cell volume regulation, and so on. 3.3 Abnormal pH related disease Most of intracellular events occur with the participation of hydrogen ions. Thus, maintenance of intracellular pH plays a key role in regulating normal cellular metabolisms. Although change of intracellular pH is rather small, its effect may be significant and even decisive. A slight abnormality in pH could lead to severe cellular dysfunction and causes diseases. Inflammatory tissues are often accompanied by a decrease in intracellular pH.44 Many neurological diseases, such as ischemic stroke, traumatic brain injury, epilepsy, Parkinson's disease, especially neurodegenerative diseases, Alzheimer's disease, are also associated with intracellular acidification.58 Ibarreta's study indicated that Epstein-Bart-transformed lymphocytes of Alzheimer's patients were more likely being induced to acidification than normal cells, and the response of diseased cells to decreased intracellular pH was slower.59 The intracellular acidification and the increased Ca2+ concentration of the neuronal cells may lead to cell apoptosis.59 In addition to neurological diseases, the abnormal pH of cancer cells has also attracted a wide attention. Due to aggressive proliferation of cancer cells, a large amount of energy input is required, and a gradient O2 tension is formed. Abnormally active cell metabolisms allow cancer cells to produce much more acid, which make the extracellular pH of cancer cells lower than that of normal cells, in the range of 6.7-

50,60 7.1. Because of the O2 tension and the lack of adequate blood vessels, the discharged protons accumulate in the extracellular matrix and produce an acidic microenvironment. Acidic extracellular environment improves the activity of related proteases, facilitating the migration and invasion of cancer cells. Given the intracellular acid accumulation may induce apoptosis, blocking the efflux of H+ may be a way to treat cancer.60,61 Although H+ is a very simple ion, abnormal pH is critical to the pathology study, diagnosis and treatment of a variety of diseases. Therefore, accurate measurement and regulation of cellular pH gains more and more attention, and gradually plays a more significant role in many applications. 4. General consideration in intracellular pH sensing The increasing ability of precisely manipulating materials structures at nanoscale leads to fast development of various nanomaterials for intracellular study. The fast emerging of new nanomaterials, on one hand, brings different physicochemical properties. On the other hand, however, these new properties put enormous challenges in full understanding the interactions between nanomaterials and biological matters. Nowadays, nanomaterials can be produced in various sizes, shapes and a wide range of tunable compositions. A comprehensive understanding of influences of nanomaterials physicochemical feature on their biological properties, including but not limited to biocompatibility, cellular uptake, transportation, biological fate, etc, is highly needed. Numerous studies have attempted to design and utilize nanomaterials for intracellular pH sensing. Intracellular pH sensing requires nanomaterials should have the following features: (1) suitable physicochemical properties, such as size, shape composition, and biodegradation; (2) excellent interfacial characters, including biocompatibility, cytotoxicity, and protein adsorption; (3) and appropriate sensing capability, including measurement range, sensitivity, response time and stability. Although various nanomaterials have been continuously used for intracellular pH sensing, a significant knowledge gap still exists on building a complete profile of nanomaterials for intracellular pH sensing. Most published studies on intracellular pH sensing lacks information on the methodology from a biological point of view, making further improvement slowly and without a clear guidance. Thus, it is imperative to develop a full spectrum of factors which should be taken into consideration to guide the design of new nanomaterial and to optimize the utility of nanomaterials for accurate intracellular pH sensing. 4.1 Biocompatibility, cytotoxicity, protein adsorption For intracellular pH sensing, biocompatibility is one of key properties that nanomaterials must have. Without biocompatibility, sensors are not safe for biological use and are not able to report the correct pH values without influencing normal cellular activities. Since nanomaterials are widely used in the commercial, biomedical and environmental sectors, even in sunscreens, toothpastes and food additives. Intensive discussions on the potential influences of nanomaterials on human health and environment must be under way. Therefore, cytotoxicity of nanomaterials have raised great concerns before their biological applications. Cytotoxicity of nanomaterials is depending on not only the physicochemical properties of nanomaterials, but also on the type and intrinsic biological properties of cells. Although lots of efforts have been devoted to reveal their relationship, there is no common knowledge to predict the cytotoxicity of certain materials towards a kind of cells. The cytotoxicity is a biological outcome from complex interaction of nanomaterials with cellular molecules and structures. Before reaching the exterior membrane of a cell, nanomaterials firstly feel the microenvironment of extracellular space, and interact with proteins and other biomolecules in the extracellular matrix. The absorption of biomolecules on the surface of nanomaterials forms the so-called “protein corona”,62 which is recognized as the biological identity of nanomaterials. The composition of protein corona is not fixed over time, and keeps changing within their biological fate, because the morphology of protein corona is considered as a loose network of proteins. The formation of protein corona can significantly change the surface properties of nanomaterials, therefore strongly influence their cellular uptake and intracellular fate. According to the binding strength and protein exchange rate, the protein corona can be divided into the “hard” and “soft” corona. It was reported that the soft corona coated nanoparticles have lower cellular uptake efficiency than that of hard corona coated or uncoated nanoparticles.63 This also give an efficient way to improve bioavailability of nanomaterials by modifying proteins onto their surface. Ge et al. showed that the carbon nanotubes (CNTs) with serum protein can disperse more evenly, and they can easily enter into a series of cells without causing any apparent cytotoxicity.64 Therefore, by properly modification of proteins onto nanomaterials surface, the chemical properties of nanomaterials can be tuned to a better state to achieve intracellular pH sensing. In most cases, the formation of protein corona should be prevented, since the absorbed protein will completely block the designed surface functionalities of nanomaterials. This is mostly done by surface modification of nanomaterials with long- chain poly(ethyl glycol) (PEG) or developing a zwitterion structure on the surface of nanomaterials.65-68 The surface properties, such as coating, ligand, composition, charge and wettability, definitely influence the interactions with membranes, organelles, ions, etc. This may affect the structures and functionalities of biomolecules and cells, leading to homeostasis destruction or induction of toxicity. The cell membrane consists of an anionic hydrophilic outer surface, so cationic nanomaterials are more easily taken up than neutral or anionic ones.69 Therefore, increasing the surface cationic charge is an effective way to promote cellular up-take. Xia et al proved that cellular uptake of cationic PEI (polyethyleneimine)-coated MSNP (mesoporous silica nanoparticles) is much easier than unmodified MSNP (silanol surface).70 Longer length polymers with a higher density of cationic surface groups exhibit better ability of attaching to negatively charged membrane than shorter length polymers. However, high cationic density could cause intracellular calcium flux and cytotoxicity, leading to physical membrane damage.70,71 It has been proved that cationic polystyrene nanoparticles with amine-functionalized surfaces have relationship with a massive macrophage cell death following lysosomal rupture, intracellular calcium flux and mitochondrial injury.71,72 This can be explained by the proton sponge effect.73 When particles pass through cell membranes, enter into an acidifying lysosomal compartment, the unsaturated amino groups are capable of sequestering protons supplied by the v- ATPase (proton pump). This process produces redundant Cl- ion and water molecule, thereby leading to osmotic swelling and lysosomal rupture, following particle deposition in the cytoplasm and the spillage of the lysosomal content. However, by adjusting polymers size, the cytotoxic effect of higher molecular-weight polymer can be reduced.71,74 Therefore, careful control of cationic density and size can facilitate intracellular uptake with little or no cytotoxicity. For dissolvable nanomaterials (e.g., quantum dots, copper nanoparticles, iron oxide), the dynamic dissolution process is affected by solvent properties such as pH, ionic strength and concentration, thus may differ from various cellular compartment. If the dissolution process takes too long, it may result in decrease of enzyme or proton pump activity, leads to overload of nanomaterial, and end up with cell death. What’s more, the free ions released by dissolvable nanomaterials (eg. quantum dots core degradation) may induce heavy metal toxicity. These problems could be solved by modification of the outer surface, or forming a core-shell structure. Li et al.75 demonstrated that engineered nanoparticles may contribute to pulmonary morbidity or allergic inflammation through the elicitation of an oxidative stress mechanism. In order to evaluate nanoparticles cellular toxicity, Jeng and Swanson et al.76 have studied the effect of different types of metal nanoparticles on the cellular function, as well as the cellular proliferation and biological end-points. Metal oxide nanoparticles used in the study included TiO2, ZnO, Fe3O4, Al2O3, and CrO3 with different particles size. The result showed that the toxicity effect is depend on the concentration and chemical composition of nanoparticles. The flow cytometry analysis showed that ZnO (50 to 100 μg/ml) can cause significant mitochondrial dysfunction and lactate dehydrogenase leakage in a dose-dependent way, while Fe3O4, Al2O3, and

TiO2 had no measurable effect when their concentrations are below 200 μg/mL. It is notable that even at high concentration, the toxicity of Al2O3 was moderate, there was only slight toxicity of Fe3O4 and TiO2 and no toxicity of CrO3 was observed. 4.2 Biodegradation Ideally, nanomaterials aiming for intracellular sensing and biochemical applications should be noninvasively cleared from cells in a suitable period after completing their task. Otherwise, nanomaterials may accumulated inside cells and cause apoptosis and dysfunction. To construct a nanomaterial with low or no toxicity degradation pathways for intracellular pH sensing, the biodegradability and toxicity of the nanomaterial itself or the toxicity of degraded products should be all taken into consideration. Some nanomaterials, such as biodegradable polymer and peptide, could be degraded in hydrolytic way by cellular enzymes. For example, poly(D,L-lactide- coglycolide) (PLGA) and polylactide (PLA) can be hydrolytic degraded in cells and the metabolic products (e.g., lactic acid, glycolic acid) could get into biocompatible metabolic pathways. These materials are safe to use and are already approved for medical use by the Food and Drug Administration of United States. Shah and Amiji77 have developed an intracellular saquinavir (an anti-HIV protease inhibitor) delivery system by using poly(ethylene oxide)-modified poly(epsilon-caprolactone) (PEO-PCL) nanoparticles. PCL is a biodegradable polymer, showing excellent biocompatibility and neutral biodegradation products, which do not affect the pH balance of the degradation medium. In intracellular pH sensing, it is crucial that nanomaterials themselves or the biodegradation products do not change the local pH. Except being cleared by the mononuclear phagocytic system in a living model, self-destructive nanomaterials have been engineered. Park et al.78 presented luminescent porous silica nanoparticles (LPSiNPs) with intrinsic near-infrared photoluminescence enables imaging cells. LPSiNPs show self-destruction in a short time with no measureable toxicity. Thus, the controllable rates of self-destruction can be considered as a desirable design criterion for the assessment of nanomaterials biodegradability. 4.3 Size and shape Recent researches have revealed that the size and the shape of nanomaterials strongly influence their cellular uptake, transportation and accumulation. In order to improve cellular uptake efficiency, great efforts have been devoted to precisely tune the size (for zero-dimensional nanomaterials) and aspect ratio (for one-dimensional nanomaterials) of nanomaterials. The thickness of most membrane bilayers is 4-10 nm. In general, there are three ways for nanomaterials cellular entry: phagocytic pathways, pinocytotic pathways and direct penetration. The selection of entry pathway of nanomaterials is restricted by the corresponding portal dynamic and size rules.79 For instance, ligand-modified nanoparticles with diameter approximately 120 nm are endocytosed mainly through clathrin-mediated pathway. For those nanoparticles larger than 120 nm, it is unlikely to enter the cell by clathrin-mediated endocytosis (shown in Figure 5). Therefore, before designing the nanomaterials, it is important to know some innate biology rules.

American Chemical Society.

Chaudhuri et al. have found out that the attractive or repulsive interactions between nanoparticles have remarkable effects on the cellular uptake characteristics.80 In the absence of interactions between nanoparticles, nanoparticles with intermediate sizes showed best uptake behavior. In a case of existing interactions, attractive interactions would increase the optimal uptake, while repulsive interactions would produce a more symmetric distribution and reduce the optimal uptake. Li and Schneider81 provided a quantitative evaluation about the cellular uptake process of gold nanoparticles (AuNPs) with different sizes (15, 30, 50 and 80nm). Results showed that 50 and 80 nm AuNPs are taken up to a greater extent than smaller size particles due to the effect of sedimentation. However, the taken up rate of smaller particles is much faster in the first 10 h. Kettler et al.82 demonstrated that decreasing the size of silver nanoparticles (AgNPs) could increase cellular uptake, and the AgNPs uptake could be improved in the medium without fetal calf serum. From these two examples above, we can find out that no universal law that governs the relationship between particle size and cellular uptake. The influence of particle size on their cellular uptake differs from case to case, which not only depends on the properties of particles, their aggregation, but also on the cell type and cultivating media. Except for size, the aspect ratio of nanomaterial is another important parameter that could determine the rate of cellular uptake, transportation, the mechanism of uptake and biocompatibility. He and Park83 demonstrated that particles with higher aspect ratios are more likely to adhere to the cells and be internalized by cancer cells. Meng et al.84constructed a mesoporous silica nanoparticles library composed of nanoparticles with different aspect ratio (AR) to study the effect of AR on cellular uptake in Hela and A549 cancer cells. They demonstrated that the rod-shaped particles with intermediary AR (2.1-2.5) were taken up in a larger number compared to shorter or longer length rods by a micropinocytosis process, which also exhibited better drug delivery ability. Qiu et al.69 observed that gold nanorods form small aggregates in culture medium and then enter the cell in the aggregated form. Longer rods tend to form larger aggregates, thus longer rods may be more difficult to internalized with cell membrane since more energy is needed for internalization. Liu et al.85 found that the rod-like bionanoparticles with various aspect ratio had different internalization pathways in different cells. By changing the aspect ratio, the entry method may also change thus leading to unexpected influence on cellular uptake rate and efficiency. 4.4 pKa of Indicators The selection of a proper indicator is the key to precisely measure intracellular pH, since different indicator has distinct pKa and measurement range. The indicator should be protonated or deprotonated at the site of interests to generate a measurable signal. There is a distinct different between the pronated form and deprotonated form of indicators, and the most sensitive region of the measurement is described by the pKa of the indicator. When the pH is at the pKa value, the indicator will present the highest sensitivity for pH measuring, and a slight change in pH will generate a significantly large measurable signal. As mentioned above, the intracellular pH in different compartment or different regions of cells varies a lot. Thus, it is important to select the right indicator with its pKa matches the pH of the interest. When the pKa of the selected indicator is more close to the pH to be measured, the measurement will surely be more accurate. In this case, the pKa of indicators plays an important role in the selection of dyes for a special application. In the past decades, a lot of pH-sensitive indicators have been synthesized. Especially the development of fluorescent pH indicators greatly simplifies intracellular pH measurement, since these indicators will have distinct fluorescence properties in their protonated and deprotonated form. Fluorescein and its derivatives are widely-used pH indicators since they are easy and cheap to prepare. The pH indicator, 2′,7′-Bis-(2- carboxyethyl)-5-(and-6-)carboxyfluorescein (BCECF), was introduced for measuring intracellular pH in 1982, was considered one of the most widely used pH indicator. The pKa of BCECF is 7.0 which is ideal for sensing cytosolic pH. BCPCF, 2′,7′-bis-(2- carboxypropyl)-5-(and-6-)-carboxyfluorescein, is a homolog of BCECF, which is better for ratiometric dual-excitation due to a stronger absorbance at 454 nm. Han and Burgess43 listed the useful photophysical properties of most widely used pH indicators(Table 1). Figure 6 gives a spectrum of the pH-sensitive ranges of most widely used cellular pH stains. More importantly, researchers nowadays have mastered the technique to precisely tune the pKa of a specific indicator by modifying its chemical structure.86-88 A fruitful toolbox has been developed to synthesize indicators with different spectral properties, pKa values as well as abilities of active-targeting.86-88 By using these tools, optimized pH sensor could be developed according to the specification of application. With the help of nanoparticles, the apparent pKa of obtained nanosensors could be tuned by loading dyes with different pKa. Peng et al.89 have reported the first ratiometric fluorescent hydrogel nanoparticles (nanogel) which is capable of sensing pH from 6 to 8. The nanogel was made pH-sensitive and fluorescent by loading pH indicator bromothymol blue (BTB) along with two standard fluorophores into the biocompatible polyurethane polymer. Li et al.90 have synthesized a series of ratiometric near-infrared (NIR) fluorescent probes which owns a stable NIR hemicyanine skeleton with different substituents. Among the series, NIR-Ratio-BTZ has an ideal pKa value (around 7.2) and the largest emission shift with the pH change, which made it significantly suitable for the minor physiological pH fluctuation monitoring and high resolution pH imaging in live cells. Koji et al.91 have synthesized indocyanine green (ICG) derivatives with nucleophilic substituents as pH-resonsive near-infrared dyes. Among the ICG derivatives, two pH-responsive dyes with pKa at 6.5 and 5 responded to the acidic intracellular compartments of HeLa cells, thus they are considered proper for high- contrast optical imaging of acidic compartment in living cells or acidic sites in vivo.

λmax,abs λmax,em Dual Exc or Indicator pKa φ Ref (nm) (nm) Em Oregon Green 0.97 (pH 9 92 490 514 4.8 excitation 488 buffer) CDCF 503 525 4.7 excitation NA 93 C.SNARF-4Fa 520 582 6.4 Both NA 94 HPTSa 405 514 7.3 excitation 1.0 (pH 5.5) 95,96 HPTSb 465 514 1.0 (pH 9.0) ACMA 419 484 8.6 NA 0.66 (pH 7.2) 97 pHrodot 560 585 6.5 NA NA 98 (a acidic or phenolic form; b Basic or phenolate form, NA: not applicable) 4.5 Measurement range The measurement range of certain intracellular pH sensors is restricted by the factors like the pKa of dyes, the pH-sensitive ranges of the pH indicators, and the properties of nanomaterials for carrying the dyes. It should be mentioned that every pH indicator only has a limited measurement range, centered at its pKa. There is no indicator that having broad pH-sensitive range to cover the whole pH range. Sensors with broad pH-sensitive range are needed when one would like to study the distribution of pH inside cells or in different cellular organelle. To widen the pH sensitive range, researchers have developed nanomaterials doped with more than one pH-sensitive indicators. Such method requires accurate control over the ratios of the dyes and their distribution in the nanomaterials. S. Chen et al.99 has reported a pH-sensitive fluorogen which can respond sensitively to pH in the entire physiological range. The dye, in essence, is a tetraphenylethene-cyanine adduct (TPE-Cy). TPE-Cy can show red or blue fluorescence in the acidic and basic environment. The Klimant group100-104 has presented a series of novel NIR-emitting aza-BODIPY pH indicators which cover the pH scale from 1.5 to 13. All synthesized dyes show excellent spectral and photophysical properties, and are representative building blocks for the development of pH sensors with extended dynamic ranges. However, in most cases, a broad pH-sensitive range is not necessary. In the case to study the pH variation in the existence of certain stimulus, a pH sensor with very narrow but shape response to pH is highly required. Such sensor possesses extremely high sensitivity, which could reveal tiny pH variation under stimulation. A breakthrough was made by the group of Gao,105 who designed a library of ultra-sensitive polymer nanomaterials to measure pH. The pKa of the polymer nanomaterial could be precisely tuned in the range of 4.4 to 7.1 with 0.3 pH increment, and the materials exhibit very sharp response to pH change within 0.25 pH unit. This kind of ultra-sensitive materials for pH is very useful for intracellular studies. 4.6 Response time The pH at different regions in a cell is not constant over time, which is maintained in an equilibrium by cellular metabolism. When the rate of acidification is equal to that of acid consumption, the pH of a cell will reach a relatively steady state. The acidification rate represents the effects of metabolic acid production, and many molecules are associated with protonation and deprotonation of H+. The consumption of H+ during the metabolic reactions would remove acid, leading to pH increase. When

- the cell is moved from CO2 free environment to a place containing CO2, the cell would meet with an immediate acidification due to CO2 influx. A new steady-state pH that is lower than the original one will be established. Thus, it is important to shorten the response time of an intracellular pH sensor in order to capture the fast pH-changing events. Normally, the response time of typical pH fluorescent sensors is a few seconds or longer. The pH sensors with response time of about a few seconds are considered to have a fast response towards pH. It is not easy to shorten the response time, since the response time is limited by protonation rate of indicators, diffusion of H+ into the sensing medium, and the size and shape of nanomaterials. The use of nanomaterials as carrier to locate the pH-sensitive dye on their surface not only increases the local concentration of pH indicators, but also facilitate proton diffusion, both of which are favorable for the improvement of response time. 4.7 Stability The stability of intracellular pH sensors can be divided into two parts: the photostability of indicators and the structural stability of nanomaterials. The structural stability usually refers to the chemical structural stability and dye leakage. Sensors should maintain structural stability within the measurement time range. In other words, sensors must be biocompatible, not be degraded in a short time and the different parts of the sensor should be firmly bonded together. The dye leakage from the cells is a common obstacle faced by many pH sensors, which restricted their use in living systems. Immobilization of pH indicators through covalent bond can significantly reduce dye leakage. Rates of dye leakage from cells have connection with the net charge on the dyes. Dyes with higher charge are more difficult to be expelled through cell membrane. Besides, biomolecule or nanomaterial conjugates (eg, BCECF-dextran) can effectively solve the leakage problem. Since the biocompatibility of the conjugates is improved, it is unfavorable for dyes to move from the inside to the outside of the cells. What’s more, the leakage problem can be circumvented by modification of functional groups onto the dyes. HPTS 58 (pH indicator) are more likely to remain inside cells because it has three sulfonate groups. Photo-degradation is a general character of fluorescent dyes. Photo-degradation would decrease fluorescence intensity, leading to erroneous results. However, photo- degradation can be strongly reduced by decreasing the intensity of excitation light, exciting in pulse mode, or synthesizing highly-stable dyes. What’s more, the photostability of dyes would be significantly improved by forming nanoparticles. For instance, nanoparticles prepared by Huang’s group106 through a nano-precipitation process have showed greatly enhanced photostability in comparison to physical blends of dye/polymer complexes in longtime cell tracing. Fu et al.107 have studied and compared the optical properties of Cy5 molecules in the absence and the presence of silver nanostructures. The results showed that forming nanoparticle complexes significantly restrain Cy5 blinking, reduce photobleaching and increase photostability. Sensors with good sensitivity, reversibility and high resistance to photobleaching are suited for long-term cell tracking and intracellular pH monitoring. For instance, colloidal luminescent mercaptoacetic acid capped CdSe/ZnSe/ZnS quantum dots, which owns great resistance to photobleaching, are applied to sensing pH in human ovarian cancers. 108 5. Nanomaterials for intracellular pH sensing and Imaging Since Richard Feynman, who received the Nobel Prize in physics in 1965, introduced the concept of nanotechnology, the rapid development of nanomaterials in the last three decades have made great changes not only in research, but also in industry and in our daily life. Nanomaterials are usually defined as the size of materials at least in one dimension is in nanoscale (typically smaller than 100 nm). The special physical and chemical properties of nanomaterials, including enhanced surface plasmon resonance small-size effect,109 surface and interface effect, quantum size effect, quantum tunneling effect, etc, make them highly different from their corresponding bulky materials. These unique features of nanomaterials lead to wide applications in many fields, such as catalysts,110 early-stage disease diagnosis, cosmetics, food quality control, environmental monitoring, and homeland security protection. Nanomaterials are widely explored for their application in biological research, especially for intracellular sensing. Their compact size makes them ideal material to study intracellular events without disturbing normal cellular activity. The intracellular pH involves in various physiological and pathophysiological processes, which is also considered as an important parameter in early diagnosis of diseases. As depicted in Figure 7, the pH value in different organelles varies111. In terms of intercellular pH sensing, many techniques have been developed, these including NMR, 112 microelectrodes and fluorescence imaging. The merit and disadvantages of these techniques have been summarized in section 2. Among these techniques, the fluorescence based intracellular pH sensors have attracted tremendous attention owing to their high sensitivity, compact size down to nanometer, fast response, tunable spectral and sensing properties, and capability of high throughput imaging with high temporal and spatial resolution.

5.2 Basic requirement of nanomaterials for intracellular pH sensing and imaging According to Section 4, there are basic requirements to design nanosensors for intracellular pH measurement. Firstly, nanomaterials should have good biocompatibility (low or no cytotoxicity) and are pH-sensitive, which can be used for studying the pH variation inside cells without disturbing normal cell activities. Secondly, their surface should be functionalized in order to facilitate intracellular uptake. Thirdly, nanosensors must have adequate sensitivity and optimized spectral feature to measure the small variation during cellular metabolism. Finally, they should be biodegradable after complete their task, and cleared from cells. In the following subsection, we will summarize the recent progress of using nanomaterials for intracellular pH sensing and imaging. Most of these nanomaterials are constructed via the chemical coupling of pH-sensitive dye on nano-size materials. Other nanomaterials have intrinsic pH response, and can be directly used for intracellular pH measurement. 5.3 Varies of nanomaterials for pH sensing and imaging There are two kinds of nanomaterials used for intracellular pH sensing and imaging. One is the dye-doped nanomaterials, on which surface are modified with pH-sensitive fluorescent dyes, and the nanomaterials mainly act as the carrier for the dyes. In this case, the nanomaterials themselves should be inert to pH change. The most commonly used pH-sensitive fluorescent dyes are summarized recently,86-88 which include several classes of dyes, fluorescein and its derivatives, boron-dipyrromethene dyes, Pyranine(HPTS), seminaphthorhodafluor(SNARF)/seminaphthofluorescein (SNAFL) dyes.113,43,114,115 Although these dyes have good water-solubility, they still have limitations when applied to intracellular sensing directly. Most of these dyes possess large conjugate system in their chemical structure, and tend to aggregate or dispersed in apolar region once uptaken by cells. By modifying the dyes on biocompatible and stable nanomaterials to form nanosensors, they can disperse well inside cells. Moreover, multiple dyes can be attached to or embedded in one nanomaterial, which not only dramatically improves brightness, and stability, but also can extend the pH response range and enhance signal readout. Other features such as targeting ability, encoding technology and intracellular location manipulation using magnetic beads can also be integrated with the pH sensing technology to form mult-functional nanomaterials. Up to now, varies of nanomaterials have been studied and explored, such as silica nanoparticle, semiconductor quantum dots, carbon materials, polymer nanoparticles, mental nanoclusters, proteins and so on. There merits and disadvantages in building pH nanosensors are discussed in details below. The other kind of nanomaterials have intrinsic pH response, and most of them are synthetic polymers. These polymer nanomaterials have unique features that their physical morphology varies with pH changes. Once the amine groups in the polymer chains become pronated, the generated positive charge will induce the polymer morphology changes. In most cases, solvatochromic dyes are labeled or embedded in polymer chains, and the morphology change further promotes the microenvironment changes around the solvatochromic dyes, which generate fluorescence signals for readout. On this occasion, pH-sensitive dyes are not needed, and the pH response characteristic is highly depending on the structural and pronation properties of the polymer chain. This configuration has unique properties that the pH response can be tuned by introducing different functional groups, and the sensors may possess extremely high sensitivity as well illustrated at the end of the section.

5.3.1 Silica Nanoparticles Silica nanoparticles are the mostly used nanomaterials to build pH nanosensor. Silica nanoparticles are optically transparent, low toxicity, inert to pH, (some are) degradable, and more attractively, their surface can be easily functionalized via the well-established silane technology. The pH-sensitive dyes are, in most cases, chemically bonded on the surface of silica nanoparticles, which is beneficial for protonation and reducing diffusion barrier. In order to avoid signal interferences, reference dyes that are inert to pH change are always incorporated inside or on the surface of silica nanoparticles, which will allow for ratiometric readout. The Burns group designed a ratiometric pH nanosensor116, the reference dye (TRITC) sequestered in the core coated by a sensor-dye-rich (FITC) shell, and successfully proving its intracellular sensing capability in rat basophilic leukemia mast cells (RBL-2H3). Because of the mature silane technology and nanodimensional silica coating techniques, other features, such as location manipulation using magnetic particles, active-targeting capability, local heating and treatment, and control drug releases, can be easily intergraded along with pH sensing. Tian et al developed a mitochondria-targeted single

•- fluorescent probe for simultaneous sensing and imaging of pH and O2 in mitochondria.117 At the same time, the rapid development of silica technology makes silica nanoparticles with different size and surface functionalities are readily available with affordable price. However, they are still not the perfect nanomaterial for intracellular pH sensing, because the remained surface dangling bonds during synthesis and surface modification will not only lead to poor biocompatibility, but also may influence the apparent pKa of the bonded dyes.108 A list of typical silica based pH nanosensors and their spectral and pH responses are summarized in Table 2.

Table 2. Typical silica based intracellular pH sensor. Name of pH Diameters Ratio- pKa Cell type Ref. sensor indicators (nm) metric FITC&RITC@ FITC/RITC 50 Yes 6.5 Hela cells 118 MSN FITC- FITC/ QDs 56±5 yes 6.5 mitochondr 117 SiO2@QDs ia core/shell silica FITC/TRIT 70 yes 6.4 RBL-2H3 116 nanoparticles C cells 119 Cy-PIP@SiO2 tricarbocyan 88.5 No 7.1 Hela cells ine R6G-FITC- R6G/FITC 110 yes - lysosome 120 MSNs

5.3.2 Quantum dots Quantum dots (QDs) have raised great attentions due to several advantages. Firstly, their narrow emission and broad excitation ensure the capability of being an ideal donor to fluorescent probe. The Bao team showed a novel FRET-based sensor for pH.121 The sensor was constructed by immobilizing pH-sensitive fluorescent proteins (FPs) on a bright and photostable semiconductor QDs. It combines the advantages of QDs and FPs, forming a FRET pair which can exhibit a >12-fold change between pH 6 and 8. Secondly, QDs have superior photostability compared with other luminescent materials, which allow for long-term pH monitoring. However, the degradation paths of QDs in cells are a challenge that should not be ignored. Except for silicone QDs which have relatively good biocompatibility in cells, most QDs are found to have significant potential risks to cells. The most widely used QDs typically contain toxic materials, such as cadmium, and lead, which put great threaten to normal cells metabolism. To solve this problem, core-shell structures are developed to overcome the leakage of toxic ions. The core-shell structure can not only enhanced luminescent properties of QDs, but also reduce the leakage of toxic materials. However, it still necessary to study the stability of core-shell structure at intracellular condition. 5.3.3 Carbon dots (including Graphene QDs) Among QDs, carbon dots are a rising star in pH sensing application, mainly due to their strong fluorescence, nontoxicity, nonblinking property and good solubility. Because the extensive resources of carbon, the production cost of carbon dots are much lowered. They have been broadly applied in the photocatalysis, drug delivery and of course, nanosensors.122 Thanks to their small size and good compatibility, pH sensors based on carbon dots have good intracellular dispersity. They are more evenly distributed inside cells, rather than accumulating in some specific organelles.123 Carbon dots themselves can be used as pH sensors. Wang et al reported a green approach for the preparation of carbon dots with dehydrated shiitake mushroom, which can be applied as an intracellular pH sensor in the range of pH 4.0-8.0.124 Figure 8 shows a label-free carbon dots (7.9 nm) as ratiometric fluorescence pH nanoprobes.125 This material displays a good linear relationship between luminescence intensity and pH in the range of 5.2-8.8 without using pH indicators. The label-free carbon dots can overcome the drawbacks of dye photobleaching and avoid the complex modification process, which can be a new promising material for intracellular pH sensing. Of course, carbon dots can also be labelled with pH sensitive dyes for ratiometric pH sensing. Shi et al reported the first example of carbon dots based tunable ratiometric pH sensor 123. The pH-sensitive fluorescein isothiocyanate (FITC) and pH-insensitive rhodamine B isothiocyanate (RBITC) were labeled on the amino-carbon dots, yielding the dual- labeled carbon dots. By varying the feed ratio of FITC/RBITC(from 1:1~1:30), the pH sensitive range could be tuned from 5.2-8.5, which fits most intracellular pH ranges. Song et al synthesized a kind of tunable pH sensor by doping carbon dots with rare earth ions, which forms a ratiometric pH sensor.126 With the pH value rising, the luminescence intensity of Eu3+ greatly increased when excited at 365 nm. 127 There is no widely accepted luminescence mechanism for carbon dots.128 It is reported that some kind of surface functional groups will introduce new energy level for electron transitions, which will greatly affect emission properties of carbon dots129. In 2011, Guo et al reported a simple, rapid and eco-friendly carbon dots-based pH sensor.130 It featured excitation-dependent on-off or off-on pH signaling, which display a linear relationship when pH is in range of 4.5-8.3.

Figure 8. Schematic Diagram for the Preparation of Label Free Carbon Dots and

Their Application for Intracellular pH Sensing19. Copyright © 1997, American Chemical Society. The graphene quantum dots(GQDs) are important member in carbon dots family, which consists of a single atomic layer of nano-sized graphite. Similar with carbon dots, GQDs possess characteristics both of graphene and carbon dots, and have been widely applied in the field of bioimaging, drug delivery, DNA cleavage, photocatalyst.131 However, an unequivocal photoluminescence mechanism of GQDs is still absent, the current mechanisms include many factors, such as size, shape, edge effect, surface structure, etc.132 The GQDs are also used for pH sensing, Huang et al fabricated a kind of N-doped GQDs, which display pH-sensitive behavior in a wide range from pH 2 to 9.133 In addition, GQDs can also be used in combination with other materials for more powerful sensing. Kim et al reported a block copolymer-integrated GQDs, It can simultaneous sense temperature and pH, as well as dose-dependent responses to different types of metal ions with excellent reversibility and stability. In addition, Kim group presented a pH sensor based on graphene oxide.134 The graphene oxide sheet is connected with blue and orange color-emitting QDs by a polymer linker. Under varying pH stimulation, the linker polymers can change their conformation, and this will influence the efficiency of FRET between graphene oxide sheets and QDs, which can indicate the pH by QDs fluorescence. The pH sensor exhibited excellent reversibility in aqueous media, which is an important requirement for intracellular pH sensing. 5.3.3 Upconversion Nanoparticles Upconversion nanoparticles (UCNPs) exhibit obvious advantages compared to conventional stokes-shifted luminescence probes. They can be excited at longer wavelength in the near infrared region using a cheap continuous laser source, and they emits in the visible. This unique feature makes them good candidates for intracellular sensing and imaging, since the longer excitation will have much deeper tissue penetration, lower autofluorescence background, and severe tissue damage by short- wave and intense radiation will be avoided. However, when lanthanide-doped UCNPs are applied in bioanalysis, biomedicine, and imaging, there are many challenges that have to be taken into consideration, such as toxicity of heavy metals, poor capability of cellular uptake and difficulties of surface modification. To the best of our knowledge, most of UCNPs are coated with hydrophobic capping ligands135, which make them insoluble and unstable in aqueous solution, and are difficult to be directly used for intracellular studies. The ligand exchanging technique provides a solution to this problem. Tuomas et al

3+ 3+ described an improved pH nanoprobes based on NaYF4:Yb , Er particles coated with polyethylenimine,136 and pH-sensitive rhodamine dyes can be covalently attached to the probe (Figure 9). An upconversion resonance energy transfer system was formed, which has a broad responsive range from pH 8 to 4, with a pKa around 6.5. Another solution is to coat the UCNPs surface with hydrophilic materials, such as silica. Kong et al developed a self ratiometric luminescence nanoprobe based on förster resonant energy transfer (FRET).137 In this system, the pH-sensitive ﬂuorescein isothiocyanate (FITC) and upconversion nanoparticles (UCNPs) were served as energy acceptor and donor, respectively. Another similar example is from Arppe et al, who reported a kind of upconversion pH nanosensor.138 This nanosensor was based on the resonance energy

3+ 3+ transfer from hexagonal NaYF4:Yb , Er nanocrystals to the pH-sensitive fluorophore pHrodo Red. In order to coupling the pHrodo Red NHS ester, the nanocrystals were coated with a thin shell of aminosilane.

5.3.4 Polymers 5.3.4.1 Synthetic polymer Polymer materials are a kind of important materials that developed rapidly in biological applications. Synthetic polymer materials have many attractive properties, such as monodispersity, biocompatibility, controlled composition and chain length, and tunable chemical properties. Moreover, Synthetic polymers are readily available with abundant species and multiple functional groups, which not only reduces the difficulty in modifying different pH fluorophores, but also offers multiple options to choose proper dyes. Most of these synthetic polymer materials do not have inherent pH response unless they are specially designed for pH sensing, so they mainly used as carriers for pH-sensitive dyes. Albertazzi et al designed three kinds of dendrimer-based fluorescent nanosensors for measuring pH in living HeLa cells. 112 As shown in Figure 10, three different pH-sensitive dye are modified along with a reference dye on the dendrimers to form the pH nanosensors, which allows ratiometric imaging of intracellular pH. Søndergaard group described the design and application of a polyacrylamide-based nanosensors (~60 nm)139 for pH sensing, The polymer was labelled with three pH sensitive dyes, fluorescein and Oregon Green, and the pH- insensitive fluorophore rhodamine. The sensor exhibits a pH measurement range from 3.1 to 7.0, and has been successfully applied in measuring endosomal or lysosomal pH. The bulky polymers can be simply converted into nanosize particles via the precipitation approach.140-142 The pH-sensitive dyes can be labelled on the polymer chain either before or after nanoparticle formation. With the development of nanotechnology, polymer nanoparticles can also be prepared via the wet-chemistry, which are start form monomers, and formed nanoparticles with uniform size.

Figure 10. The dendrimer-based fluorescent pH sensors112 © 2016 Elsevier B.V. All rights reserved. 5.3.4.2 Natural polymer Compared with synthetic polymers, there are many polymers existing in nature, such as polylysine, chitosan, lignin, starch and so on. The major advantages of natural polymers are that they are nontoxic, renewable, storage rich, low price, biodegradable and have high biosafety. Aylott et al labelled cellulose nanocrystals prepared from cotton with pH-sensitive fluorophores to form pH nanosensors.143 Chiu et al used an associating polyelectrolyte N-palmitoyl chitosan to develop a FRET-based dual emissive pH sensor,144 which are successfully applied in measuring pH 7.5-4.0 intracellularly or extracellularly. However, the choice of natural polymers is still much less than synthetic ones, which limits their application in pH sensing. 5.3.4.3 Ultra-sensitive polymer Most of currently reported pH nanosensors are based dye-labelled nanomaterials. Their pH responses are highly limited to the pKa of the dye itself. The pH measurement range spans several pH units, which makes them have relatively low sensitivity. In most cases, the pH values inside cells, especially in certain cellular locations, are maintained almost constantly in order to keep normal cellular activities. Small variation in intracellular pH always related with cellular dysfunction and diseases, which requires pH sensors must have extremely high sensitivity. However, most of currently developed pH sensors cannot fulfill this requirement. A breakthrough was made by Gao’s group, who reported a kind of ultra-sensitive stimuli responsive nanomaterials for pH sensing and imaging.145,146,147,148 Tertiary amino groups were introduced in the polymer chains and act as ionizable groups, which can protonate at a different pH.146 At physiological pH, the neutral tertiary amino groups self-assemble into the hydrophobic cores of micelles, resulting in aggregation of fluorophores, and leading to the quenching of fluorescence. At this stage, the micelles exhibits very weak fluorescence despite being full loaded with fluorophores (Figure 6). On the contrary, when the pH value decreases below the transition value, amines groups become protonated and the polymer chain are positively charged, which leads to micelle disassembly. The disruption of micelles releases fluorophores to the surroundings, and cause dramatically increases of fluorescence emission. The ultra-sensitive pH sensors exhibit a very sharp pH response and generate a large fluorescence intensity change. For example, during the pH changing from 7.4 to 6.7, one kind of nanosensors showed a 102-fold increase in fluorescence intensity. These sensors have extremely high sensitivity, which can differentiate at least 0.25 pH changes.108 This kind of high pH sensitivity makes them have the capability to differentiate tumors from normal tissues easily. Moreover, because the tertiary amine groups on diverse polymers (such as different chain length of liner polymers or different ring size of cyclic polymers) have the different transition pH values. By precisely controlling the transition pH value of tertiary amine groups, the pH response and the pKa of resulted nanosensors can be tuned to match application requirement. Gao and colleagues have set up a library of ultra-sensitive pH nanosensors consisting of 10 components with 0.3 pH increment that span the entire physiologic range of pH (4-7.4) .105 Those exquisite sensitive probes make a great contribution to the development of tumor imaging.

5.3.5 Macromoleculars Biomacromolecules have significant advantages when employed as pH sensors, because they have excellent biocompatibility, and easy and safe biodegradation. However, cautions much be taken since the fluorophore-labelled biomolecules become new kind of materials, and their cytotoxicity much be test before use. Biomolecules are not widely used as pH sensors because they have relative high price, and the labelling and purification processes are quite complicated, and are time- and labor-consuming.125 There are proteins that are inherently fluorescent and can be genetically modified and expressed as pH sensors. Aliye et al developed a number of green fluorescent protein (GFP) mutants via protein engineering approach,149 and found the fluorescence and absorbance properties of various GFP mutant proteins were strongly pH dependent. Yellen’s group engineered the first ratiometric, single-protein based red fluorescent sensor of pH (pHred) via mutagenesis of a red fluorescent protein mKeima150. The pHred sensor was used to image intracellular pH in live cell with an apparent pKa of 6.6. Moreover, they also found that the fluorescence lifetime of pHred is pH dependent, suggesting it can be used to image intracellular pH via fluorescence lifetime imaging microscopy (FLIM) technique. The FLIM technique has excellent precision in intracellular sensing since it can eliminate the impact from cellular autofluorescence and is independent on fluorophore concentration. Poëa-Guyon et al took advantage of the strong pH response of enhanced cyan fluorescent protein and demonstrated its application as a pH sensor. Schmitt employed the pH-sensitive GFP derivative and FLIM to simultaneously measure pH in cytoplasm and mitochondria.151 6. Perspectives The rapid and continuous innovation in nanotechnology provide a fruitful library of nanomaterials for biological applications. Nanomaterials possess unique properties which are not provided by their corresponding bulky ones. Their compact size, large surface-to-area ratio, ease of functionalization and outstanding architecture at nanodimensional make them ideal materials for intracellular studies. The advances in nanomaterials also push the intracellular pH sensing on to a higher level. Thanks to the nanotechnology, many pH nanosensors with superior sensing properties, represented by the ultra-sensitive pH nanosensors based on polymer micelles (Section 5.3.4.3), were designed and fabricated. However, there is still a need of breakthrough in intracellular pH sensing, since current sensors do not have good photostability. They are not suitable for long-term monitoring intracellular pH. Innovation in signal generating and readout technology are highly needed, which is significantly reduce the influences of dye photobleaching. In addition, although lots of efforts have been devoted on synthesizing dyes with suitable pKa for intracellular study, much of knowledge is still unknown how to precisely control dye pKa by modifying their chemical structures. There is a clear trend that researchers are trying to understand the mechanism of controlling dye pKa and photostability. With the fast accumulations of knowledge, it is believed that people will master the technique in precisely controlling these parameters. Another trend in pH sensing is that pH sensors are always used along with other sensors. Our group has devoted lots of effort in designing and fabricating multiple sensors for simultaneously measuring multiple parameters inside cells. By using multiple sensors, fewer nanomaterials will be introduced inside cells, but more parameters will be recorded using these sensors. The use of multiple sensors could induce less perturbation to normal cellular activities, and multiple intracellular parameters can be monitored not only at the same time, but also at the very same site, which are very beneficial to study certain intracellular event.

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