Lateral flow assay: a promising rapid point-of-care testing tool for infections and non-communicable diseases

The POCT is a rapidly growing field of clinical diagnostics that is expected to be one of the primary drivers of the future in vitro diagnostic industry. It is an auxiliary, bedside, and decentralized testing, often done at the site of patient care [7]. This technique does not require a specific premise to carry out the tests as they can be done at primary care settings, for instance, at doctors’ workplaces, emergency units, intensive care, and other medical clinic units, operation rooms, schools, working environments, homes, and military operation areas [15]. Glucose, cardiovascular, and pregnancy tests are the main aspects that govern the vast majority of the point-of-care (POC) diagnoses in the current medical line globally [3].

Even though POCT had a slow reception when first introduced in the 1990s, the use of POCT later expanded massively due to the rise in consumers’ need for self-testing and improvement in medical technology. Today, various POCT devices and instruments are available in the market that can serve in various fields [7, 16]. They have become an essential detection strategy in different clinical settings to diagnose diseases depending on their properties and ability to recognize many types of analytes such as metabolites, proteins, and microorganisms [15]. Different properties of various POCT devices are explained in Table 1.

The POC-geospatial concepts are widely applied by many countries across the globe as a precaution against disaster, for potential risk awareness and management, and as communal health adversaries such as outbreaks. Integration of both the concepts helps respond quickly and control the spread of deadly outbreaks such as Ebola, Middle East respiratory syndrome-based coronavirus, and malaria. Many healthcare systems based on this concept are being employed in works to be implemented in countries with a high-risk potential of outbreaks. Termed as geospatial-POC preparedness, the ideology of preparing a mobile healthcare facility with improved and easy-to-handle diagnostic tests can improve the management of world countries with respect to outbreaks and border security [19].

The POCT tools are highly dynamic due to their advantages in diagnosing a wide range of infectious and non-communicable diseases (NCDs). This is because various types of samples can be used as the target material for conducting detection and analysis. Interestingly, several POC tests have been developed, focusing on the principal infectious disease-causing microbes, such as malaria parasites, human immunodeficiency virus (HIV), human papillomavirus (HPV), dengue, Ebola, Zika viruses, and Mycobacterium tuberculosis (TB) bacteria [20]. For instance, rapid diagnostic test lateral flow strips capable of detecting malaria parasite proteins have been used for the early diagnosis of malaria in tropical areas [21], enabling prompt artemisinin-combined therapy (ACT) [22]. The development of a POCT tool that can quantify CD4+ T-lymphocytes and HIV viral load for HIV detection [23], POC test platforms that can replace visual inspection with acetic acid (VIA) for HPV diagnosis [24], and the formulation of the ReEBOV antigen rapid test kit for the rapid detection of the Ebola virus in East African countries [25] provides additional supporting evidence for the use of POCT in the context of infectious diseases.

At the same time, the increase in morbidity and mortality rates in recent times due to alarming levels of NCDs such as diabetes, stroke, cancer, type-2 diabetes, and Alzheimer's disease has exponentially increased the demand for POCT applications for diagnosing these diseases [26]. The diagnosis of NCDs, which previously heavily relied upon laborious biochemical measurements, is now being replaced by simpler detection tools without compromising on detection sensitivity and specificity [27]. Recent research has reported the use of POCT to screen for individuals suffering from hypertension and obesity, improving the potential to manage these conditions while raising awareness about NCDs [28]. The diagnostic strategies for cancer have also changed their trajectory toward POCT, which previously relied upon conventional biological tests that may take up to few weeks to complete. The immunoassay-based POCT facilitates the early detection of several cancer types such as pancreatic cancer or prostate cancer, only requiring less volume of body fluid or blood sample [29]. At the same time, the use of POCT also helps in the early diagnosis of diabetes by analyzing HbA1c [30], further cementing the improved role of POCT in noninfectious diseases.

Given the current epidemic situation caused by SARS-CoV-2, POCT has emerged victorious among the other conventional diagnostic principles such as reverse transcriptase-polymerase chain reaction (RT-PCR) [31]. This is due to the ability of POCT to offer rapid diagnosis and accurate and timely detection, and the tests can be carried out at the user's end, thus exponentially increasing the rate of diagnosis in a short time [32]. The rapid test kits for SARS-CoV-2 detection are readily available in pharmacies and healthcare facilities, and the public can get them at a very low cost to perform their detection test.

The POCT devices are mainly designed based on membrane-based microfluidic technology. This is because other materials such as glass, silicon, or polymers would require a complex designing mechanism involving extensive processes and additional instruments. This would increase the overall manufacturing expenditure of the devices, making them not compatible with POCT. Papers, on the other hand, are easy to fabricate and do not require specific cutting mechanisms and are regularly used for most of the POCT devices design. Thus, these devices are also known as paper-based analytical devices [33].

Cellulose and nitrocellulose are the two main kinds of membranes used for POCT device development and manufacturing [34]. Cellulose is a linear macromolecule made up of an array of glucose units. They are organic solvents and insoluble in water. Filter paper and chromatography paper are examples of cellulose-based paper materials and are often used for dipstick assays and microfluidic devices (μPADs) [17]. On the other hand, a partial nitration process is carried out to produce nitrocellulose, where this process converts the hydrophilic property of cellulose into a hydrophobic one. Lateral flow devices (LFDs) are mostly designed using this material [33]. Both cellulose and nitrocellulose are porous materials, and nitration improves the porosity of the materials, granting a slight advantage to nitrocellulose over cellulose. The porosity shows the wetness of the material and determines the flow of liquid on the membrane. Moreover, the surface chemistry of these membranes is also given special attention due to its influence on particle immobilization, adsorption, and colorimetric changes during the assay process [34].

POCT devices usually come with a simple design, where most of them are available in pure disposable form. They are formatted for single-use and can detect antigens or antibodies from a wide array of specimens. Lateral flow and immunochromatographic strips (ICS) are examples of such devices. Due to their simple nature, the production cost is very low, and they are inexpensive compared to permanently integrated device strategies such as polymerase chain reaction (PCR). Moreover, these devices are affordable in most of the developing countries with resource-limited settings [35]. In some cases, the disposables are integrated with a reader to get the reading of the analyte detected, such as found in a blood glucose analyzer [35, 36].

On the other hand, these testing devices are designed to be lightweight, portable, and user friendly such that they can perform the complex tests at the site where they are most needed [7]. They can provide detection results instantly due to limited or no sample processing steps that accelerate the testing process. Hence, raw samples such as urine or blood can be used as primary samples for analyte detection [15].

Before POCT, detection strategies heavily relied on complex techniques such as microtiter plate reader devices. An electrical power source was needed to operate the devices, making them non-portable and the tests needed to be conducted in laboratories. However, with the introduction of POCT that primarily uses immunoassay technologies, detection can be easily done with maximum sensitivity. The use of immunoassay techniques also helps in coping with the need for rapid turnaround times of the test. There are three assay techniques such as agglutination, immunofiltration, and immunochromatographic assays [7].

A biosensor is designed on the principle of analyzing a biomolecule quantitatively and converting the result into a visible signal [41]. With the current trend of exploiting the science of nanotechnology becoming widespread, the adaptation of this technology in the POCT approach enabled the development of nanobiosensors as advanced bioanalytical devices [38, 39].

Nanobionsensors are no different from any of the other kinds of biosensors or bioanalytical devices available. In fact, they are used for the same purpose, which is to quantify the biochemical or any biological event by the means of electrical, optical, or magnetic technology. However, unlike other conventional biosensors, nanobiosensors use a compact probe that is derived from various nanomaterials [40, 41]. The nanomaterials used can show their novel characteristics better compared to the original materials that do not have nanoscale features. Several nanomaterials have been used for the biosensing devices such as nanotubes, nanorods, nanoparticles, and nano-thin films [42].

Nanobiosensors overcome most of the limitations identified in conventional biosensors that became a barrier to expanding their use in various fields. This is because the nanoparticle size and shape can be optimized during synthesis to reach the maximum electrochemical active sites [43]. The nanomaterials are also designed to provide high sensitivity for detection; hence, they can locate the analytes from the sample and eventually provide outcomes with maximum precision [43]. In cases where noble metal nanoparticles such as colloidal gold nanoparticles (AuNPs) are used, optical sensing can be vastly improved by enhancing the surface plasmon resonance (SPR). Reducing the SPR intensity of the nanoparticles allows them to collect more light from the surroundings due to their optical sensitivity, and signals with great intensity can be produced [44].

As of today, several forms of nanobiosensors are available and used in various fields such as biomedicine, optics, catalysis, and electronics. However, affinity-based nanobiosensors that are available in the simplest form possible remain atop of the others. In other words, LFAs are the most-preferred nanobiosensing tools, operating based on the affinity of the biorecognition molecule to the sample and forming a complex [45].

Lateral flow assays (LFAs)

The LFAs are the paper-based POCT biosensors that are primarily used for the detection and quantification of a specific target from a complex mixture [46]. They come as preassembled strips of a transporter material encasing dry reagents that are activated by applying the sample of interest in liquid form and the eventual result can be observed from 5 min to 30 min [47]. LFAs are accountable for single use at the POC [48] such as when used to determine pregnancy, the collapse of inner organs, the pathogenic effect of specific microorganisms including biowarfare agents, a toxic mixture in consumables, feed and environment, and illicit drug abuse [47].

A typical LFA consists of an application pad, conjugate pad, nitrocellulose membrane, and adsorption pad mounted on plastic backing [49]. The pads and membrane are generally porous. The pores in the sample pad can be distributed symmetrically or asymmetrically and they help to filter any coarse materials from the sample before reaching subsequent sections of the instrument [48]. The whole assay works depending on the wettability and capillary flow action; hence, the pore walls need to be wetted by the sample liquid. The sample is dropped onto the sample pad after mixing with viscosity enhancers or buffer salts that enhance the sample viscosity and improve the overall flow rate [50]. Cellulose filters and cross-linked silica are two common materials used to manufacture the pads, and the selection of the material heavily relies on the objectives of the test and characteristics of the sample [45, 51].

In the conjugate pad, labelled conjugate elements have been applied and dried during the manufacturing process. When the buffer-diluted sample is applied to the sample pad, it migrates to the conjugate pad via capillary action and dissolves the conjugate element in the liquid. The interaction between the sample and the conjugate element results in the sample-label tagged conjugate complex formation, which is imminent for the final reaction at the membrane. By filling the pores in the pad, the newly formed complex can migrate to the reaction membrane [48].

Reaction membrane is an important part of the LFA instrument. Very often, the reaction membrane is designed using nitrocellulose due to its porosity. Nylon, polyethylene, and fused silica are a few new materials that have been used to design in some of the recent research. The affinity of the sample, such as a protein, with the porous structure is very important for the interaction with the capture probes available in the test line and control line [52]. The test line probe and control line probe are immobilized onto the membrane to be recognized by the complex. The colorimetric changes at the test line depending on the presence and absence of the analyte of interest determine the reaction, while a signal at the control line validates the assay [46]. The components of the LFA strip are shown in Figure 2.

Figure 2.

The sample pad is the proximal end of the strip where the sample is placed for a test run. This is the area where the sample is also treated. The treated sample will flow through the strip to the conjugate pad and form a conjugate with label-tagged conjugate probes. Normally, conjugate particles that are used in LFA have unique optical, electronic, and/or structural properties such as AuNPs and up-conversion nanoparticles. The analyte then migrates to the reaction matrix membrane or more commonly known as nitrocellulose membrane where a control line and a test line are fabricated by depositing control probes and capture probes. Nitrocellulose membrane is the main material in LFAs as it grants a stage for reaction and detection during the assay. At the end of the strip, the wick or absorbent pad collects the excess reagent from the membrane.

In the past decade, the use of LFAs has been expanded to various fields, which simultaneously skyrocketed the need for them, especially where rapid tests have become prevalent. This is because these diagnostic devices can emulate the criteria gazetted by the World Health Organization (WHO) that specifies the requirement for an ideal POC device. The criteria are known as the “ASSURED test” where the diagnostic tools should be affordable, sensitive and specific, user-friendly, rapid and robust, equipment-free, and deliverable [53]. LFAs come with a simple designated structure, making them portable and equipment-free. The manufacturing cost is very low compared to other diagnostic tools; hence, LFAs are available in the market at an affordable range. The assays can also be performed at the patients’ site of care by anyone without needing any complex procedure or skilled personnel. Thus, LFAs are widely regarded as ideal POC devices and are preferred the most by consumers and regulatory authorities [46]. The different sensing modalities and advantages and disadvantages of LFAs have been explained in Table 2.

Table 2.

Sensing modality, features, advantages, and disadvantages of various LFAs

Sensing modality	Features	Advantages	Disadvantages	References
Optical	Colorimetric	Colorimetric changes help to make a visual observation for result interpretation	Sensitivity issues that lead to result inaccuracy	[53]
	Fluorescence	Provides results with higher signal to noise ratio; higher sensitivity	Fluorescent labels are not visible at low concentration; requires external reader for specific analysis	[54]
	SERS	High sensitivity with very low detection limit; can be developed into ultiplex LFA for a wide range of detection	Expensive	[55]
	Chemiluminescence	High sensitivity when used together with metal nanoparticles; lower LOD; highly stable for immobilization	Requires addition of enzymatic substrate; low shelf-life	[56]
Thermal	Thermal imaging	Improved LOD and high sensitivity; no photo bleaching or photo instability	The infrared image sensors are massive; expensive	[57]
	Laser speckle imaging	Uses improved depth resolution reader that improves sensitivity	Expensive and not suitable at resource-limited settings	[58]
	PA imaging	PA imaging helps sample penetration deeper that improves the sensitivity of detection	Strips need to be dried before analysis since acoustic waves travelling through water-interface is a challenge	[44, 59]
Magnetic	MPQ	Low detection limit and highly sensitive	Not suitable for multiplex detection when using high affinity variants	[52, 60]
Electrochemical	Amperometry, cyclic voltametric, impedimetric	The ratio of applied voltage can be adjusted to increase the sensitivity of the detection; nanoparticle integration to improve LOD	Requires expensive reagents and multistep procedure	[44, 61]

LFA, lateral flow assay; LOD, limit of detection; MPQ, magnetic particles quantification; PA, photoacoustic; SERS, surface enhanced Raman scattering.

Lateral flow assay design

LFA protocols can be divided into two groups, i.e., competitive (inhibition) and noncompetitive (sandwich) assays [62]. The mechanisms of competitive and noncompetitive assays are shown in Figure 3.

Figure 3.

In a sandwich assay, the analyte with multiple determinant sites from the sample reacts and hybridizes with the conjugate particles to produce analyte–conjugate complexes at the conjugate region. The complexes now migrate along the membrane via capillary force. Once the complexes reach the test line, they interact with the immobilized capturing molecules that are complementary to one of the determinant sites, forming a conjugate–analyte–capture molecule complex. The analyte is sandwiched between the conjugate and capture molecules. The complex results in a strong visible signal formation. Free conjugate particles move past the test line and migrate to the control line. At the control line, another capturing molecule complementary to the conjugate probe interacts and forms a signal, showing LFA validation. In the absence of an analyte, no complex is formed at the test line and no signal is produced [62].

Competitive assays are suitable for testing small analytes with single determinant sites and those that cannot bind to two antibodies simultaneously. In the presence of analytes in the sample, the analytes compete with the capturing molecules for binding sites at the test line. As a result, there is no aggregation of conjugated particles at the test line; hence, no visible signal is produced and a positive result is received. When there is an absence of analyte, capturing molecules at both the test and control lines will react with the conjugated particles and produce a visible signal [63].

Besides the improved diagnosis rate, cost-effectiveness also drives the implementation of LFA-based diagnosis across various medical sectors. As discussed earlier in this review, rapid test kits for SARS-CoV-2 were not only cheaper but also easily accessible to the public, enabling instant testing and screening for the infection. This helped reduce the reliance on laboratory-based testing methods like RT-PCR, which, in addition to being laborious, incurred high costs in terms of personnel, machinery, and testing facilities [64].

Furthermore, cryptococcal antigen (CRAG)-LFA was used to screen for cryptococcal meningitis (CM) among HIV patients in Uganda. CM served as a major cause of morbidity and mortality (approximately 10%–44%) among HIV patients. The use of CRAG-LFA in this resource-limited setting maximized disease diagnosis capacity and highlighted the need for additional research methods. Importantly, the use of such POCT reduced the burden on the country to establish laboratory-oriented diagnostic facilities while maintaining cost-effectiveness in line with their health economic principles[65].

In another report, it was concluded that the use of POCT in hospital EDs significantly contributes to cost management compared to using conventional diagnostic methods, further emphasizing their importance in cost-effectiveness [66]. However, it is worth noting that the cost of POCT can fluctuate, sometimes exceeding that of traditional methods, depending on the clinical setting, target condition, and test quality [67].

LFAs, which are primarily based on antigen–antibody interactions, have shifted their paradigm toward using nucleic acid sequences as the sample for the detection of amplicons with the aid of PCR [68]. This allows LFAs to be used in medical conditions that cannot be diagnosed precisely using antigens and antibodies, but oligonucleotides are required, instead. Apart from this, new labels such as quantum dots and upconverting phosphors are used to improve the sensitivity of LFAs to detect analytes even at lower concentrations [69]. Nonmetallic nanoparticles such as carbon nanotubes (CNTs) and graphene oxides are now been significantly integrated with LFAS, producing CNT-based colorimetric LFAs [46]. These LFAs have been shown to produce remarkable sensitivity as that of the AuNP-mediated assays, suggesting these nonmetallic nanoparticles can replace the metallic nanoparticles in the future. In terms of improving the detection signal, enzymes like horseradish peroxidase (HRP) are used where they help to magnify the signal intensity by helping assembly of detection particles around the sample, and the chemiluminescence signal is measured using specific tools [70]. Along with chemiluminescent detection, fluorescence-based detection strategies are also widely used in LFA technology because they have lower detection limits and help in quantitative analysis [71].

Apart from optical detection, magnetic nanoparticles (MNPs) are used as labels for detection. They employ the magnetic particles quantification (MPQ) technique to quantitatively evaluate the result obtained [53]. A stray magnetic field is generated via external magnetic forces and the intensity of the magnetic field around the MNPs is quantified using the MPQ technique [72].

At the same time, few LFA detection strategies have been discovered to circumvent sensitivity problems such as using thermal response to quantify the analyte concentration in the sample. These thermal imaging–based LFAs use infrared cameras or laser speckle to detect the temperature distribution around the LFA strip under the illumination by photothermal (PT) excitation light. This PT-based thermal imaging method helps to evaluate the improvisation in the limit of detection (LOD) and sensitivity [73].

Moreover, surface-enhanced Raman scattering (SERS) LFAs are now being widely used for the detection of the influenza virus [55]. It is a surface-sensitive–based detection where the target molecules are attached to the SERS tracers; upon reaction, a Raman scattering spectrum is observed and measured using Raman spectrometer [58]. It still employs AuNPs as reporter probes and is regarded as more sensitive than conventional LFAs and hemagglutination assays [71].

Reducing the scale of the LFAs and optimization of those devices to make them even more convenient and portable is an important goal aspired to be achieved in the future [53]. Prospects like integrating LFAs in lab-on-a-chip design are underway to extend their use for more resource-limited settings [74]. Moreover, researchers also want to introduce LFAs with micro electro-mechanical systems and improve their sensory transmissions so that devices like mobile phones can be used to operate the assay or their use can be translated even in military service [53].

On the other hand, multiplex LFAs have been introduced to carry out analysis on more than a single analyte using the same test. Due to the increasing trend in discovering new biomarkers, the conventional individual biomarkers detection using multiple LFA strips is no longer an ideal approach. It is necessary to detect multiple biomarkers at once in order to produce information that is insightful or definitive in contemporary clinical settings. Hence, researchers started normalizing using multiplex LFAs that can detect all the biomarkers using a single strip, and the result was remarkable [76]. Several test lines were designed on a single strip and targets were detected via spatial resolution. This approach is often used in the food industry [75]. Multiplex LFAs, while retaining the integrity of a single LFA system, increase the detection sensitivity and reduce the overall cost for multiple tests. This helps integrate the reader system with the multiplex LFAs that can improve the result analysis while maintaining the cost at a rate suitable for low-resource settings [77].

Researchers’ interest in nucleic acid-based detection became key for the invention of various biosensors and bioanalytical devices that employ nucleic acid sequences as primary samples [78]. Known as nucleic acid biosensors (NABs), they are useful for clinical diagnostics of inherited diseases and pathogenic infections [79]. One of the most promising NABs that are currently being used regularly is NALFAs.

NALFAs are paper-based analytical devices designed based on the similar structural framework of conventional LFAs that use nucleic acid samples or modified aptamers as recognition molecules [34]. To date, they are not just used for clinical diagnosis of diseases caused by pathogenic microbes, but also expanded to various other fields of research including plant pathology, veterinary diagnostic, food safety, and environmental diagnostics [80].

The assays can be divided into two formats. The first one involves the hybridization of single-stranded amplicons (ssamplicons) to complementary probes that are immobilized onto the membrane. The second format involves specific-tagged double-stranded amplicons (ds-amplicons) coupled with an antigen–antibody reaction. This format is known as nucleic acid lateral flow immunoassay (NALFIA) and requires PCR amplification of amplicons before assay [47]. Nevertheless, both formats serve a single purpose, which is to generate rapid, effective, and highly sensitive test results.

POC devices that can detect the variations in human genetic sequences are still under development and have not been generalized like other device types [81]. Researchers came very close a few times in fabricating fully functional NALFAs that can employ nucleic acid sequences to detect the nucleotide mismatches and mutations present in the sample [82]. For example, a multidisciplinary research team of the National University of Singapore (NUS) developed enVision, a device used for screening of infectious diseases using the enzyme-assisted nanocomplexes for visual detection of nucleic acids, where this device was tested for the detection of cancers markers and human genetic markers [83]. A research team developed an amplification refractory mutation system–lateral flow assay (ARMS–LFA) to study the individual genetic variations via single nucleotide polymorphisms for detection purposes and subsequently treating the genetic disorder [81]. These researches gave promising early output but still it requires improvisation. Nevertheless, these attempts have now paved the way for advanced research in developing NALFAs for human genetic disorders diagnosis.

PCR-based techniques were extensively used for microbial-based disease detection to understand the genetics of the microorganisms, especially, the RNA viruses [84]. Currently, SARS-CoV-2 caused by an enveloped spike-like coronavirus from the genus Betacoronavirus has succumbed the whole world in one of the deadliest pandemics in history, spreading to almost every country in the world and causing millions of deaths to date. This is because the genomic RNA of the viruses instantly comprehends the hosts’ cellular machinery and is translated into protein, necessitating the use of PCR technology such as reverse transcription-quantitative polymerase chain reaction (RT-qPCR) for systemic detection [85].

However, qPCR was not the ideal method for the long-term detection strategy, considering the need for highly skilled personnel, centralized medical facilities, and sophisticated sample-preparation technology with automated systems. This eventually increases the overall expenditure needed to carry out the tests and would not be suitable for countries with low medical facility settings [86].

At the same time, POCT tools such as NALFAs were slowly being introduced in most medical settings, suggesting an easy way for detection. They come with great specificity but the available POCTs could not replicate the sensitivity that the nucleic acid amplification could offer [85]. Hence, the amplification strategy was revised, and an isothermal nucleic acid amplification was discovered as a proper solution to all those issues faced earlier [87]. Isothermal nucleic acid amplification is the process of effectively accumulating nucleic acid sequences at constant temperatures [88]. This amplification technique was later combined with a lateral flow system, forming isothermal nucleic acid amplification–based NALFAs [89]. This intervention allowed researchers to progress even nearer to producing devices that satisfy the ASSURED strategy [90]. This isothermal amplification is also expanded into several different formats.

Recombinase polymerase amplification-LFA (RPA-LFA) is a highly sensitive nucleic acid amplification technique, employing recombinase complex from T4 bacteriophage to introduce primers to specific DNA sites and begins amplification by the strand displacing DNA polymerase in the presence of adenosine triphosphate molecules [86]. It is often regarded as one of the best alternatives to PCR and believed to have subdued imperfections found in other isothermal approaches such as nucleic acid sequence-based amplification (NASBA), strand displacement amplification, rolling circle amplification, the loop-mediated isothermal amplification (LAMP), and helicase-dependent amplification [91].

RPA-LFA eliminates the template denaturation step, which is important for the PCR technique. There is no temperature fluctuation needed as this technique can operate at a low and constant temperature ranging from 38 °C to 42 °C [92]. Recombinase polymerase amplification (RPA) only requires about 10 copies of template strands for amplification, showing its greatly improved sensitivity matching with the PCR technique [84]. In one of the recent studies, it was mentioned that RPA can still produce amplification signals notably within 10 min even at the near-detection-limit template concentrations, reassuring the capability of this amplification technique as a great alternative and to be combined with lateral flow detection [86].

The RPA-LFA combination first came into light nearly a decade ago and is used for the discovery of many microbial species such as HIV-144, Canine Visceral Leishmaniasis, Orientia tsutsugamushi, Rickettsia typhi, plasmodium, intestinal protozoa, cryptosporidiosis, yellow fever virus, Penaeus stylirostris, Plasmodium falciparum, Entamoeba histolytica, Schistosoma haematobium, little cherry virus, and plum pox virus [91].

The combination allowed to increase the LOD of many NALFA formats and eventually quickened the detection process [93]. Moreover, tailed primers are often associated with RPA, anchoring for the improvised hybridization capacity of the amplicons. The use of tailed primers also surpasses the need for hapten labeling and use of capture and reporter antibodies and terminates postamplification processing [91]. The entire assay, including amplification and detection, could be completed within 15 min through all the modifications, showing the efficiency of RPA and rapid hybridization kinetics of the tailed amplicon with the surface-immobilized probe and the reporter probe and making them an effective choice for fast, low-cost and highly sensitive NALFAs [91].

NASBA is a transcription-based amplification and a part of the isothermal nucleic acid amplification, exclusively designed for the detection of RNA targets [81, 83]. This technique is also known as “self-sustained sequence replication” due to its capability of generating new copies of nucleic acid template sequences with or without denaturation process, the same as RPA [94]. An abundance of antisense RNA copies are produced at the end of the DNA NASBA and is usually recognized by the hybridization of specific fluorescent molecular beacons [16].

NASBA is often associated with oligochromatographic detection using specific dipsticks and is deemed an indispensable contribution in countries with poorly equipped laboratories. The amplified product is applied and allowed to migrate on the sensitized membrane of an oligochromatographic stick. Capture and detector probes functionalized with nanoparticles then hybridized with the amplified product because of migration and produced a signal as an indicator [94].

LFA will detect the amplified RNA product quantitatively. This in turn enables researchers to analyze samples with lower concentrations because NASBA can amplify the sample and eventually maximize the LOD of LFA. On the other hand, NASBA-LFAs undergo optimization including oligonucleotide concentration, buffer composition, membrane flow rate, strip size, and nanoparticle size to keep the overall cost of the LFAs as low as possible even though amplification is required. The signal produced by the NASBA-LFAs will be analyzed based on signal-to-background ratio calculation since a stereo microscope equipped with a color camera is used for nanoparticle capture analysis. LFAs with the best resolution show they can detect NASBA products, proving NASBA enables detection of short, amplified genomic sequences through LOD improvisation [93].

Recently, new work was initiated to use the NASBA technique as a testing strategy for SARS-CoV-2 Virus (COVID-19). The project was named as Isothermal NASBA-Sequencing based hIGH-throughput Test (INSIGHT). It is a two-stage COVID-19 test strategy, combining an isothermal NASBA technique with NGS. Crude saliva is employed for isothermal NASBA reaction in the first step, and billions of copies are produced within 2 h. The amplified sequence is then subjected to the NGS stage to intensify the sensitivity and specificity of the test in a scalable manner. The amplification is monitored in real-time using molecular beacon readouts, and researchers are working on the possibility of using lateral flow assay to receive rapid test outcomes [95].

LAMP technology is a novel nucleic acid amplification method that works on the principle of auto-cycling strand displacement DNA synthesis [96]. This action is being carried out by Bst DNA polymerase large fragments [97]. This technology was discovered with the same motive as other isothermal amplification techniques, which is to improve the sensitivity and sensitivity of nucleic acid amplification for detection. Since its inception in the year 2000, the technique was emulated directly into a diagnostic assay for the detection of various diseases and now remains alongside RPA and NASBA as one of the isothermal amplification techniques associated with LFAs [98].

LAMP uses 4–6 primers recognizing 6–8 distinct regions of target DNA for a highly effective amplification [96]. The reaction begins with a strand-displacing polymerase, catalyzing strand invasion of the template by hairpin-forming LAMP primers. These primers are responsible for annealing and extension and are later replaced by displacement primers to initiate amplification, forming a dumbbell-like structure [99]. This structure was retained throughout the process and became the mechanics for amplification [100].

Same as RPA, LAMP uses a single and constant temperature for amplification ranging from 60 °C to 65 °C. LAMP-based assays do not require thermocyclers and tedious operational mechanisms like PCR, making them easy to perform with advanced specificity and incorporate in point-of-need diagnostic tools such as LFAs to improve the detection strategy [96].

LAMP-LFAs are appropriate diagnostic tools to be used in various fields and are commonly used in the agricultural field to understand plant virology [101]. They easily replace the gel electrophoresis procedure that is commonly required in conventional PCR techniques to see the product. Even though they both have a similar role to a certain extent, LAMP-LFAs are better than electrophoresis because they come in as predesigned simple structures and do not require multiple steps [100]. The sample loading is a lot easier compared to electrophoresis where skilled personnel is required. Furthermore, the outcome can be visualized easily via LFAs whereas electrophoresis does not come with that luxury and often requires multiple attempts [101].

Recently, different attempts were carried out by researchers to improve the sensitivity of the LAMP-LFAs. One of those was to introduce filtration-based LAMP-LFA. In this assay, the microbial cells were concentrated by filtration instead of cell enrichment culture. A cellulose acetate filter was used to concentrate the Escherichia coli cells in this research, improving the concentration of the sample required for analysis. The genomic samples were then isothermally amplified using LAMP and visually detected by LFA. The outcome was overwhelming, but the overall process took a long time and does not suit the criteria of POCT devices [60]. In another research, LAMP-LFAs are further modified into multiplex LFA to use them for the detection of more than one sample at a time. A bacterium that has heat-stable protein resistant to protease in the gastrointestinal tract that can cause food poisoning is observed through this format. Multiplex LAMP-LFAs help to carry out multiple tests at one time using a single membrane, hence greatly reducing the need for repetitive LAMP and a sample application with different LFA kits [61]. Recently, an RT-LAMP assay was developed to detect SARS-CoV-2 in real-time without using an RNA extraction kit. The test was fast and effective and believed to be one of the alternatives to detect the presence of the virus at the patient's site without analyzing in centralized laboratories [99, 100].

Antibodies have been and are still being used as a key recognition element in assays. Monoclonal antibodies such as mouse hybridomas are widely used nowadays due to their proven affinity and specificity [46]. However, denaturation induced by temperature, time-consuming and laborious chemical modifications, and not suitable for nonimmunogenic detection makes the antibodies a vulnerable choice to be used in LFA without further modifications. As an alternative, aptamers became a preferred choice for the detection strategy due to their advantage over the areas where antibodies lack [102].

First introduced in the 1990s, aptamers are short single-stranded DNA or RNA sequences. They possess enormous binding affinity and sensitivity for molecular targets ranging from macromolecules to micromolecules [89]. Unlike antibodies, aptamers have no batch-to-batch variation, have extended shelf-life with very minimal immunogenicity, and easily get along with any chemical modifications to improve the binding capacity. Due to these advantages, aptamers were incorporated in LFAs replacing antibodies, and different types of aptamers-based LFAs (Apt-LFAs) were subjected for use in various fields such as disease diagnosis, agriculture, and food industry. Despite that, these Apt-LFAs are not commercially available yet and still fall behind antibody-based LFAs in the aspect of general use [102].

Aptamers and antibodies have similarities in terms of tertiary structure-based target recognition [89]. Hence, most Apt-LFAs are designed based on the idea of antibody-based LFAs. They are abundantly used for the sandwich assay method [103] compared to the competitive assay method due to their ability to easily recognize large molecules such as proteins. The first sandwich Apt-LFA was developed for thrombin detection using dual aptamers that can target different recognition sites of thrombin molecules. The first aptamer was conjugated to the AuNPs and loaded onto the conjugate pad, serving as reporter aptamer. The reporter aptamer also includes a short poly-A tail, which later is easily recognized by the complementary sequence with an additional poly-T tail at the control line. Then, the biotinylated second aptamer immobilized onto the test zone via streptavidin-biotin interaction, serving as capture aptamer. Both the aptamers were able to bind to different sides of the thrombin, forming a sandwich complex and eventually producing a red signal at the test zone. A red signal was also produced at the control zone because of the interaction between the unbound detection aptamer conjugate and oligonucleotide sequences at the control zone that is a complementary detection aptamer. This dual aptamer strategy was highly preferred due to its high sensitivity and unmatched success rate. In recent years, aptamer–antibody combination and split aptamer strategies were used but their applications later were ceased due to instability and low sensitivity [102].

For the competitive assay, dual aptamers are not used because naturally this type of assay is performed for smaller molecules with fewer recognition sites. In this assay, an oligonucleotide partially complementary to the aptamer sequence will be immobilized onto the test line. When target analyte is applied onto the sample pad, they migrate and eventually are bound to the aptamer–reporter complex. When the newly formed complex reaches the test line, a competition takes place between the targets in the sample and the preloaded targets upon the binding affinity toward the detection aptamer, and no signal will be produced at the test zone. If target analytes are absent in the sample, then the aptamer–reporter complex is easily detected by the preloaded target at the test line, forming a strong signal. The interaction between detection aptamer and the oligonucleotide sequence complementary to detection aptamer immobilized at control line produces a signal, validating the LFA [91].

Aptamer immobilization onto the membranes is an important part of the Apt-LFA fabrication [89]. Usually, noncovalent strategies are used to immobilize the aptamers onto the nitrocellulose membrane. Biotin and streptavidin are the important and most-used components for noncovalent attachment. The biotin molecules will be conjugated at the end of the aptamer and later incubated with membrane-immobilized streptavidin to form a strong binding between the aptamer and the membrane with high affinity [104]. The use of biotin–streptavidin interaction often results in stability issues, making them incompatible with nucleic acid aptamers. Hence, microgel-mediated immobilization, tandem repeating–mediated aptamer immobilization, and ultraviolet (UV)–based covalent binding immobilization strategies are now progressively tested for strong immobilization [102].

The POCT has completely shifted the paradigm of diagnosis strategy in today's world. The development of LFAs with greater sensitivity and specificity, while they emerged as highly cost-effective technologies, enabled fast diagnosis of many infectious diseases. At the same time, LFAs that use nucleic acid samples for detection such as NALFAs improve the detection sensitivity and duration for NCDs, further expanding the administration of POCT in various fields to replace laborious conventional methods.

Despite these advantages, the requirement for confirmatory independent tests for certain conditions, the increase in cost for qualitative analysis, and technical blunders are a few shortcomings often associated with POCT devices, particularly for LFAs. However, the recent and prospective innovations targeted upon POCT and LFA can improve the effectiveness of this technology and make it indispensable for the future.