Microfluidics and Nanofluidics (2020) 24:17 https://doi.org/10.1007/s10404-020-2321-z REVIEW Challenges and perspectives in the development of paper‑based lateral fow assays Surasak Kasetsirikul1,2 · Muhammad J. A. Shiddiky1,3 · Nam‑Trung Nguyen2 Received: 4 September 2019 / Accepted: 29 January 2020 © Springer-Verlag GmbH Germany, part of Springer Nature 2020 Abstract Lateral fow assays (LFAs) have been introduced and developed over the last half century. This technology is widely used as a tool for diagnosis in several felds such as environment, food quality and healthcare. Point-of-care (POC) diagnosis using LFAs has been attracting attention of the research community, particularly aiming for the development of a platform that can evaluate of biological markers in bodily fuids such as saliva and urine. The existence of a disease or the pregnancy can be determined by a test device, before further investigation and medical treatment. LFAs make use of a disposable test strip, which can provide diagnosis result on the spot within minutes. Thus, LFAs is a promising alternative of preliminary diagnosis for laboratory instruments that are costly, time consuming and require trained personnel. This paper includes a brief over- view of the conventional LFAs: material selection based on its roles and characteristics, working principles, fundamentals, applications, and design criteria. We mainly discuss the technical challenges in both engineering and biochemical aspects and recommends possible solutions. We identify current research trends and provide perspectives of advanced technologies for enhancing assay performance. Keywords Paper-based microfuidics · Lateral fow assays · Point-of-care diagnosis 1 Introduction dedicated and large instruments to be diagnosed, resulting in high operation cost, delay of treatment and inaccessibility, Recently, the need for low-cost medical diagnostic devices particularly in remote areas (Kasetsirikul et al. 2016; Millot has been increasing, particularly in developing countries due et al. 2017). Consequently, point-of-care (POC) diagnostic to limited facilities and lack of qualifed medical personnel device is a well-suited alternative for replacing laboratory- (Morbioli et al. 2017; Yetisen et al. 2013). The gold stand- based equipment and skilled staf for relatively simple diag- ards of medical diagnosis such as polymerase chain reaction nosis (Sajid et al. 2015). (PCR) and enzyme-linked immunosorbent assay (ELISA) The World Health Organization (WHO) provides seven are traditionally based on lab-bench protocols, well-trained major guidelines for the development of POC devices sum- personnel and well-maintained diagnostics labs (Yager et al. marised in the acronym “ASSURED”: (1) afordable: rea- 2008; Clark et al. 2016). Moreover, advanced medical diag- sonable prices for settings composed of population at risk nosis can detect a disease in the initial stage, leading to early of infection, (2) sensitivity: few false negative and lower and successful treatment. However, some diseases require limit of detection, (3) specifcity: few false positives (4) user friendly: a few days of training and ease of use, (5) rapid and robust: acceptable waiting time, long shelf lives * Nam-Trung Nguyen [email protected] without refrigerators and high throughput, (6) equipment- free: self-powered sources, on-site analysis, easily dis- 1 Queensland Micro and Nanotechnology Centre (QMNC), posable, and easy sample handling, and (7) deliverable: a Grifth University Nathan Campus, Nathan, QLD 4111, portable or hand-held device (Drain et al. 2014; Grace and Australia Zaman 2012; Mashamba-Thompson et al. 2017). Meet- 2 School of Engineering and Built Environment, Grifth ing these WHO guidelines, paper-based devices have been University Nathan Campus, Nathan, QLD 4222, Australia attracting extensive attention, especially because of their 3 School of Environment and Science, Grifth University Nathan Campus, Nathan, QLD 4111, Australia Vol.:(0123456789)1 3 17 Page 2 of 18 Microfluidics and Nanofluidics (2020) 24:17 capability to be used in rural and limited resource environ- principles as well as applications of conventional LFAs is ments (Cate et al. 2015; Jiang and Fan 2016). also included to provide fundamentals for understanding Paper-based devices are cost efective, disposable and LFAs and their design criteria. can be used with different biomolecules. Paper-based devices are especially simple because of the power-free fuid transport by capillary action (Cate et al. 2015; Hos- 2 State of the art of LFAs seini et al. 2017). Paper-based materials have been used for a range of applications since the 2nd century AD in 2.1 Materials for LFAs China (Rooz 2010; Wong et al. 2009). To date, paper has been employed in lateral fow assays (LFAs), dipsticks LFAs are portable strips assembled on a plastic backing and microfuidic paper-based analytical devices (µPAD) card, consisting of diferent parts: sample pad, conjugate (Parolo et al. 2013). LFAs were frst reported in 1956 pad, fowing membrane and absorbent pad, as shown in by Plotz and Singer, who established the fundamentals Fig. 1. The fowing membrane is the area performing the of LFAs for latex agglutination assay (Singer and Plotz test, where the pre-immobilised reagents capture the analytes 1956). Since then, the working principle of LFAs has been in the sample. elaborated more and initiated extensive applications for LFAs utilise a number of biorecognition molecules, labels rapid detection of infectious diseases (Wong et al. 2009). and detection methods toward the diagnosis applications. LFAs have rapidly grown over the last few decades for The roles and material selection criteria are discussed below. qualitative and quantitative diagnosis and became a rela- tively mature technology (Sajid et al. 2015; Yager et al. 2.1.1 Sample pad 2006). Currently, LFA market value is approximately 6.0 billion USD at a compound annual growth rate (CARG) Sample pad is the frst area to get in contact with the sample. of 7.7% and is expected to reach 8.7 billion USD by 2023 The major purpose of a sample pad is to allow and maintain (Report et al. 2018). The LFA market is mainly driven liquid sample to fow through continuously. In certain cir- by the high exposure to infectious disease throughout the cumstances, if the concentration of the analytes in the sam- world, ageing population growth, high demand of POC ple is too high, fltration or dilution is required to treat the testing and home-based LFA platform (Report et al. 2018). sample before passing it through the later parts (O’Farrell LFAs beneft from advantages such as long shelf lives— 2015). Additionally, this part can be used to pretreat sample generally of 12–24 months without fridge, low cost, broad by depositing dry reagents to adjust pH for achieving proper range of applications, small sample volume, and ease of conditions for an assay (Wong et al. 2009). Therefore, the use (Sajid et al. 2015; Wong et al. 2009). Moreover, the sample pad requires a high tensile strength when it is wet. sensitivity and specifcity are determined by fow char- If the pad fails, the sample cannot be delivered through the acteristics optimisation. It depends on membrane matrix subsequent parts and the assay cannot be used for testing. and labels for biorecognition used in the assay to provide The common materials for this section are cellulose, glass acceptable qualitative and semi-quantitative readout (Sajid fbre, rayon or modifed fltration matrices (Sajid 2015; et al. 2015; Wong et al. 2009). Wong et al. 2009; Millipore 2013). During the last decade, several published review papers have discussed the recent applications of LFAs and novel materials for enhancing their performance (Yetisen et al. 2013; Sajid et al. 2015; Bahadır and Sezgintürk 2016; Hu et al. 2014). The major trend in assay enhancement is devel- oping novel materials to express more sensitivity and speci- fcity to the target, or to have multiple functions (Chen et al. 2014; Li et al. 2012; Shen et al. 2015). In addition, sample preparation can be optimised and enhanced to control and prepare the samples before introducing them into the device (Carrell et al. 2019; O’Farrell 2015). Current challenges must be clearly identifed to better understand the limitations and to provide guidelines for assay performance improve- ment. Therefore, this review mainly focuses on challenges and provides perspectives to overcome them. The review also identifes future trends of employing advanced tech- nologies in LFAs. A brief overview on materials, working Fig. 1 Conventional lateral fow assay 1 3 Microfluidics and Nanofluidics (2020) 24:17 Page 3 of 18 17 2.1.2 Conjugate pad fowing in the membrane (Millipore 2013). In addition to physical characteristics of the membrane, the properties In LFAs, each strip test needs a label for capturing analytes of liquid fowing in the membrane also afect the capillary in a liquid sample and for generating a signal at the testing fow rate. Contact angle, viscosity and surface tension play and control lines. The label is modifed with protein linked important roles in regulating fow rate, because the interac- passively or covalently for specifc analytes existing in the tion between liquid and porous membrane is determined by liquid sample for biorecognition in this area. Substantial the capillary force. The governing equations for fuid fow research on labels used for LFAs has been performed. Exam- are discussed in the later section. ples are nanoparticles made of gold (Jawaid et al. 2013; Lee The stability of protein binding to the membrane mainly et al. 2015; Mdluli et al. 2014), carbon (Bogdanovic et al. afects assay performance. Long-term bonding of protein 2006; O’Keefe et al. 2003) and other metals (Park et al. prefers to employ the hydrophobic and hydrogen bonds 2015) or composited metals (Jiang et al. 2016; Yan et al. (Wong et al. 2009). Hence, selecting reagents for protein 2014) as well as modifed nanoparticles such as magnetic immobilisation needs careful consideration.