Applied Science and Convergence Technology 2024; 33(5): 117-125
Published online September 30, 2024
https://doi.org/10.5757/ASCT.2024.33.5.117
Copyright © The Korean Vacuum Society.
Hafsa Humaa , b , † , Maryam Shabbira , b , † , Sung Eun Seob , and Oh Seok Kwona , b , c , ∗
aDepartment of Nanoscience and Technology, Sungkyunkwan University, Suwon 16419, Republic of Korea
bSKKU Advanced Institute of Nanotechnology (SAINT), Sungkyunkwan University, Suwon 16419, Republic of Korea
cDepartment of Nano Engineering, Sungkyunkwan University, Suwon 16419, Republic of Korea
Correspondence to:oskwon79@skku.edu
†These authors equally contributed to this work.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/licenses/by-nc-nd/4.0/) which permits non-commercial use, distribution and reproduction in any medium without alteration, provided that the original work is properly cited.
Point-of-care testing (POCT) equipment has transformed healthcare management by allowing rapid and accurate diagnostics at the point of care. It enables immediate clinical decisions to be taken, leading to improved outcomes. This review highlights the advances in the field of POCT achieved by integrating molecular diagnostics with advanced bioengineering techniques in the development of POCT devices. At present, POCT devices span simple glucose meters to sophisticated handheld devices for monitoring chronic diseases and detecting infectious diseases. Traditional polymerase chain reaction (PCR) has been significantly enhanced in sensitivity and specificity in terms of POCT by the innovation of the photonic PCR and digital PCR, which gives results in minutes with improved diagnostic accuracy. Clustered regularly interspaced short palindromic repeats-based graphene field-effect transistor and bioelectronic devices developed through bio-microelectromechanical system technology meanwhile have further revolutionized rapid and precise diagnostics. Additionally, lateral flow assays (LFAs) are versatile instruments essential for addressing global health challenges in managing infectious disease outbreaks. LFAs are also suitable for resource-limited countries. This review sheds light on scientific principles, technological progress, and practical applications of POCT.
Keywords: Point-of-care testing, Polymerase chain reaction, Bio-microelectromechanical system, Graphene field-effect transistor, Lateral flow assay
Worldwide outbreaks of pathogenic microorganisms causing deadly infectious diseases have threatened public health globally in the past few years [1]. Existing technologies such as immunoassays, culturing techniques, and molecular techniques require skilled employees, sample pre-treatment, expensive tools, and centralized lab facilities, necessitating large expenditures of time and money and presenting some of the biggest disadvantages at the time of the coronavirus pandemic [2]. As a result of this, research and advances in point-of-care testing (POCT) devices with high sensitivity and selectivity have led to the development of noninvasive, rapid viral detection [3].
During the past decade, POCT technologies have become a crucial component of medicine, greatly enhancing the capacity to diagnose illnesses and determine the relevant therapy at or near the location where patients are being treated [4]. Diagnostic devices serve as a connection between centralized clinical laboratories and patient management settings. Today diagnostic devices are facilitating improved clinical decision-making, saving treatment time, and enhancing overall patient outcomes. POCT integrated with modern diagnostics has resulted in rapid detection of various diseases including infectious [5], inherited [6], and chronic diseases [7,8].
All the commercially available technologies present today are based on nucleic acid assay, which provides diagnosis through the sensitive and quick evaluation of targeted deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) sequences [9]. Polymerase chain reaction (PCR) technology is the main instrument for amplification and analysis of nucleic acid and serves as the foundation of the diagnostic research area [10,11]. Recent advancements in the technology of bioengineering, microfabrication. and PCR has led to the possibility of developing sensitive POCT devices that are easy to use, portable, and sensitive.
The incorporation of bioelectronic devices into molecular diagnostics has enhanced the performance and utility of POCT devices [12]. Miniaturized diagnostic platforms are fabricated by bio-microelectromechanical system (Bio-MEMS) process that combines microelectronic and biological components to provide quick results with enhanced sensitivity and specificity. In recent years, clustered regularly interspaced short palindromic repeats (CRISPR)-Chip [13] and a bioelectronic nose were fabricated for more specific and accurate detection of the target in a sample.
Lateral flow assay (LFA) meanwhile has found widespread application in the field of diagnostics because of its adaptability and detection of a wide range of analytes from viral antigens to tiny compounds within low target concentrations [14]. The subsequent fast progress in POCT will lead to the detection of early disease, chronic conditions, and outbreak management. This in turn will alleviate strains on health care systems worldwide and improve public health response capabilities. In cases of emergency, POCT devices can allow rapid and field diagnostics because of their versatility and mobility.
The aim of this review is to provide a comprehensive analysis of POCT devices in the healthcare field including their present status and future potential. The review is focused on the different types of POCT devices including PCR, bioelectronic devices, and LFA for the diagnostic field, as shown in Fig. 1. Finally, future perspectives and improvements that are required for improving the existing technologies are summarized.
RNA and DNA are two of the most significant biomolecules that carry genetic information in living organisms. As a result, they are utilized as vital biomarkers for the detection of a wide range of diseases, including cardiovascular disorders [15], neoplasms [16], and infectious diseases [17]. Nucleic acid testing is typically performed to address two main targets, human genomic DNA for identification of genetic diseases [18,19] and pathogen genomes for diagnosis of infectious diseases for POCT [20].
The target nucleic acid concentration in clinical samples such as in blood is often low, which often falls further below the detectable limit of most prevailing detection technologies, for instance, approximately 180 ng/mL, and this spurred the development of an amplification process. In the realm of biological research, the development of PCR brought about a profound transformation. For the first time in history, it allowed the accurate identification and production of substantial amounts of DNA. PCR has proved valuable in molecular diagnostics for oncogenes, infections, hereditary illnesses, and forensic identification. The initial objective that led to PCR discovery in 1985 was to identify mutations in the hemoglobin beta gene causing sickle cell anemia [21]. Over the past thirty years, PCR has progressed from end-point PCR to real-time PCR, and now to its current iterations, absolute quantitative digital PCR (dPCR) [22] and ultrafast photonic PCR [23]. Presently, doctors and researchers are extensively utilizing this approach for disease diagnosis, gene cloning, and sequencing for POCT. Additionally, it is suitable for performing sophisticated quantitative and genomic analyses with swiftness and precision. Figure 2 shows the different generations of PCR over the past few decades.
The first generation of PCR refers to the conventional PCR technology, which was primarily utilized for qualitative analysis. It involved amplifying the target DNA using an ordinary PCR machine by the catalytic action of polymerase enzyme and analyzing the PCR products through agarose gel electrophoresis or sequencing [24,25]. The polymerase enzyme catalyzes the formation of a complementary DNA sequence by attaching a tiny fragment (primer) to one of the DNA strands at a specific position chosen for the initiation of synthesis. Primers restrict duplication of the sequence, resulting in the amplification of a specific DNA sequence with a large number of copies [26]. Conventional PCR is often regarded as the most sensitive detection method currently available. The utility of PCR as a diagnostic tool for infectious diseases was initially demonstrated in 1987 [18] and then expanded upon in subsequent years [27,28].
In 1987, Kwok et al. [18] focused on identifying human immunodeficiency virus sequences using a new PCR technique that involved amplifying DNA in the lab with high selectivity. The steps of PCR involved denaturation for 20–30 s (94–98 °C), annealing for 20–40 s (50–65 °C), and extension for 1–2 min per kb (72 °C), repeated for 25–35 cycles to amplify DNA. Each cycle doubles the amount of target DNA, resulting in exponential amplification.
The polymerase was rendered non-functional in every denaturation step and had to be reintroduced for each PCR cycle, resulting in a laborious execution of the approach [29]. The use of Taq polymerase, derived from the thermophilic bacterium Thermophilus aquaticus, which can withstand temperatures as high as 94 °C, obviated the need for adding fresh polymerase [30]. The original version of PCR technology did, however, have a significant flaws in that it was mostly restricted to qualitative analysis and was unable to produce precise quantitative analysis that is meaningful for prognosis and diagnosis [31,32]. Furthermore, agarose gel electrophoresis’s initial nucleic acid dyes, including ethidium bromide, are extremely harmful to the environment and human body and are known to cause cancer [33]. The conventional PCR method normally takes several hours to obtain results, and the electrophoresis analysis or sequencing detection process is also timeconsuming (~ 6 h).
The second generation of PCR refers to real-time PCR technology or quantitative PCR (qPCR). The qPCR technique has been advanced by the introduction of fluorescent dyes and DNA probes into the reaction system to collect fluorescence signals for monitoring the buildup of PCR products and visualizing the progress of the reaction. The cycle threshold value of the standard curve is measured to quantify the PCR product at the end of the reaction [34]. The results thus produced are comparable and repeatable when performed under the standard operating procedures. SYBR green [35], TaqMan probe [36], fluorescein amidite (FAM) [37], carboxy-X-rhodamine (ROX) [38], and many others are commercially present for the detection of PCR products.
For the first time, RNA studies became possible by the incorporation of a reverse transcription step prior to thermal cycling in PCR, a method known as reverse transcription-qPCR (rt-qPCR). rt-qPCR is normally based on a two-enzyme protocol. The first step consists of enzyme mediated cDNA synthesis at 42–55 °C for 30–60 min implemented by the reverse transcriptase. The second step involves a subsequent amplification reaction catalyzed by DNA-dependent DNA polymerases with denaturation (94–98 °C for 10–30 s), annealing (50–65 °C for 20–40 s), and extension (72 °C for 20–60 s) for 25–40 cycles [39,40]. Fluorescent markers are incorporated to quantify DNA in real-time during each cycle and hence enable accurate quantification of DNA present after the completion of 30–40 cycles. During the era of the coronavirus pandemic, reverse transcription PCR (rt-PCR) served as the gold standard.
Kuiper et al. [41] presented a novel high-temperature rt-PCR technique employing Volcano3G polymerase for the identification of severe acute respiratory syndrome (SARS) coronavirus-2 (CoV-2) RNA in samples obtained from patients. FAM/black hole quencher1-labelled probes were immobilized with a primer and the amplification was visualized by the detection of the de-quenched FAM signal on a blue light transilluminator with [the limit of detection (LOD), n = 6] within 2 h [41].
qPCR has effectively overcome the limitations of the first generation PCR in the context of quantitative analysis. The quantitative analysis approach depends on the quality of the standard curve and degree of operation technology. The presence of PCR inhibitors and identification of low initial concentration of target DNA may present challenges by distorting test results due to alteration of the background values. An absolute result (precise quantity of target DNA) cannot be quantified due to amplification bias caused by inaccuracies in estimating the fluorescence base line. Although qPCR allows quantitative detection, the amplification efficiency in qPCR also depends on the polymerase enzyme selection, presenting another limitation [42].
In order to achieve absolute counting of target molecules, dPCR, also known as the next generation of PCR technology, dispenses nucleic acid samples into numerous independent, parallel micro-reaction units (nanoliters) with unparallelled sensitivity [43]. Third generation PCR technology is further divided into dPCR, droplet-based dPCR (dd-PCR), and chip-based dPCR (cd-PCR).
dd-PCR is a technique that utilizes small liquid droplets to perform very precise and sensitive DNA amplification and quantification. The sample flows across the microfluidic chip, creating a segmented flow [44]. In 2020, Alteri et al. [45] performed dd-PCR for the detection of SARS-CoV-2 in 55 rt-PCR negative suspected coronavirus disease 2019 (COVID-19) cases. The sample was divided into many droplets, separated by an immiscible liquid to produce an emulsion. The emulsion was gathered in a vial, followed by the execution of PCR. The resulting sample was then subjected to flow cytometry to quantify the quantity of droplets exhibiting positive PCR result [45]. Temperature cycling is performed and a fluorescent image of the droplets is taken and analyzed [46]. When compared to qPCR, dd-PCR offers the benefits of increased precision and a reduced coefficient of variation in absolute quantification [47]. PCR multiplexing is commonly performed using probe-based fluorescence, where distinct excitation colors are used for each DNA/RNA [48].
In cd-PCR, a micromachining method is used for the fabrication of microwells in the silicon chip. The target nucleic acid is injected and the temperature cycling process is carried out. This is followed by fluorescence microscopy to ascertain the quantity of wells exhibiting a positive PCR outcome by imaging the chip [49]. In a study conducted by Nykel et al. [49], microarray-based cd-PCR was fabricated on silicon wafer by a wet etching process. The PCR cycling protocol consisted of a pre-denaturation step at 95 °C for 10 min, followed by cycles of denaturation at 95 °C for 10 s and annealing at 58 °C for 40 s. A total of 45 cycles were performed, and the operation concluded with a holding time at 10 °C. Charged-coupled device photography was employed to quantify the number of positive fluorescence signals within the microchamber, which revealed a imit of detection of 10 copies/μL of SARS-CoV-2 [50]. A silicon-based microfabrication technique and the incorporation of multiplexing can be carried out by integrating a heater/sensor in each well employing an ion-sensitive field-effect transistor (FET) into cd-PCR to monitor the PCR process. This eliminated the use of a separate fluorescence imaging system required for imaging [49].
dPCR involves dividing a PCR sample into numerous (in some cases millions) smaller samples derived from the original [51]. This process allows for the digitization of the pool of DNA molecules, with each smaller sample containing either a single copy or no copies of the DNA. dPCR relies exclusively on microfluidics technology and can be categorized as either droplet-based or chip-based. For the enhancement of signals, various nanomaterials have served as promising candidates including gold nanoparticles (AuNPs), silver nanoparticles, carbon tubes, and quantum dots (QDs) [52–55]. AuNPs and nanofilms are considered to be among the most efficient enhancers owing to their reproducible synthesis, low power consumption, high heat transfer rate, and simple configuration [52]. In a study conducted by Kim et al. [56], Delta and Omicron variants of SARS-CoV-2 were distinguished by utilizing the plasmonic photothermal effect of gold nanofilms incorporated in ultrafast dPCR technology. Light emitting diode-induced activation of a gold nanofilm was accomplished for quick thermal cycling. Genomic DNA was quantified by enumerating the number of fluorescent wells using fluorescence imaging equipment, resulting in high sensitivity (LOD, n = 10 copies) within 25 min. In 2023, Kim et al. [57] developed a new photonic dPCR integrated with in situ florescence detection using a high-velocity photonic scanner. A dPCR chip was prepared with a gold film using a MEMS process and N-heterocyclic carbene was used as a linker fabricated with an anti-quenching layer, as shown in Fig. 3. SARS-CoV-2 was detected within 15 min with 96.4 % accuracy, 98.6 % specificity, and 99 % sensitivity.
Bioelectronic devices have revolutionized POCT by integrating biological systems with microelectronics to create highly sensitive diagnostic devices [58,59]. Bio-MEMS is a burgeoning field wherein bioelectronic devices have been developed [60]. These devices identify and detect volatile organic molecules (VOCs) by mimicking the sense of smell of human olfactory receptors [61,62]. They implement sensor arrays that are equipped with biomolecules to selectively interact with the target VOCs, which can be peptides, aptamers, antibodies or olfactory receptors. These biological interactions between receptor and target are transformed into electrochemical signals, resulting in accurate, specific, and rapid outcomes [63]. In medical diagnostics, a bioelectronic noses has shown potential by identifying disease biomarkers in a patient’s breath and also in environmental monitoring by detecting pollutants [64]. These devices are user-friendly and portable and are thus suitable for field applications.
Seo et al. [65] presented a noteworthy advancement in bioelectronic devices. They developed a novel diagnostic bioelectronic device that simultaneously recognizes several respiratory viruses. The platform consists of a multichannel graphene FET (GFET) device, immobilized with human receptors utilizing N-heterocyclic carbene as a linker, for the sensitive and specific detection of several respiratory viruses, as shown in Fig. 4. It detects SARS-CoV, SARS-CoV-2, and MERS-CoV viruses without requiring the sample to be pretreated in any specific way. This is achieved through the multichannel arrangement of receptors such as angiotensin-converting enzyme 2, neuropilin-1, and dipeptidyl peptidase-4, which bind to the respective spike protein of the viruses. This binding produces an electrochemical signal which indicates the presence of the respective virus. The performance of this bioelectronic nose indicates that the bioelectronics-based platform could detect much lower concentrations of respiratory viruses (i.e., as low as 10 fg/μL).
Another advanced technology in bioelectronic devices is the incorporation of a CRISPR system for the identification of RNA and DNA target from a sample [66]. In a study by Balderston et al. [67], a novel CRISPR-Chip was fabricated with a Cas9 protein variant engineered with specific 20 nucleotide-based guide RNA on a graphene substrate, eliminating the requirement for intricate amplification procedures. CRISPR-Chip comprises a liquid gated GFET, functionalized with Cas9 by using phenylboronic acid as a linker. Upon the binding of the target DNA to the CRISPR-Chip, a measurable electrical signal is generated. A CRISPR-Chip based FET was distinguished by its rapidity within just 15 min with a sensitivity of 1.7 fM [67]. Mutations involved in Duchenne muscular dystrophy were successfully detected by using CRISPR-Chip, showcasing its potential for application in medical genetic testing. CRISPR-FET was fabricated by Li et al. [13] using a Cas13a system to detect various viruses without amplification. A sophisticated sensing technique was employed to convert the signal of a large analyte into an on-chip cleavage response of an immobilized CRISPR reporter, as shown in Fig. 5. This allows signal generating events to take place within the Debye length with high sensitivity for open reading frame 1ab gene (LOD of 2.6 fM) [13].
POCT is a breakthrough diagnostic domain that offers on the spot feasibility with rapid and instant results [68]. This innovative diagnostics strategy has a significant impact on healthcare, especially in resource-poor settings where access to centralized laboratories is severely constrained. LFAs are considered excellent because they are simple, affordable, adaptable, and versatile in terms of operation [69]. In general, LFAs have many applications in clinical diagnostics, environmental monitoring, and food safety [70]. The LFA operates on the principle of immunochromatography, where a sample such as saliva, blood, or urine is applied on the sample pad to check the presence of a target analyte, which can be detected because of binding to labeled antibodies already immobilized on pad [71,72]. This simple process involves utilizing a very small quantity of a sample and shows results within a few minutes [73]. LFAs are extensively accessible and can be used without specific training or equipment, which contributes to their attractiveness.
LFAs have been utilized for the rapid detection of viral diseases such as influenza, dengue, and most recently SARS-CoV-2, demonstrating their adaptability and importance in addressing public health emergencies through timely and precise diagnostics.
Colorimetric LFA test kits have been developed to provide advanced technology for point-of-care diagnostic applications. These test kits utilize a simple paper-based platform that incorporates immunochromatographic techniques, coupled with visual color changes to detect the presence of specific analytes in a sample [74,75]. The assay relies on the movement of the sample along a test strip, where it meets antibodies conjugated to colored particles such as AuNPs and then attaches to the captured antibodies on the test line [76,77]. AuNPs have several advantages including uniform size, high stability, ease of production, low cost, and their characteristic red color due to a localized surface plasmon resonance effect allowing visual detection, making them suitable for widespread applications, especially in colorimetric LFA [78]. Typically, such assays are very easy to read without the need for complex specialized equipment; a colored line on the test line normally indicates a positive result. Colorimetric LFAs are universal and have a wide range of applications such as clinical diagnostics [79], food safety [80], and environmental monitoring [81], on the basis of being easy to use, rapid, inexpensive, and portable. It is one of the most useful diagnostic techniques that is currently used in countries with low resources and limited access to full-fledged laboratory facilities.
A study by Jang et al. [82] introduced a new LFA technology for rapid and simple detection of influenza virus. The authors utilized a LFA with loop-mediated isothermal nucleic acid amplification for the detection of influenza A and B viruses (IAV and IBV). The flu- loop mediated isothermal amplification (LAMP)-LFA platform has high specificity and sensitivity in distinguishing influenza viruses from SARS-CoV-2. The identification of influenza in this experiment was achieved by attaching streptavidin-conjugated AuNPs (AuNPs-SA) onto the test strip. Binding of biotin-labeled rt-LAMP amplicons to streptavidin on the strip is indicated by a positive test line. The control line verifies the test utilizing a binding reaction with AuNPs-SA without the target analyte.
LFA can detect dengue infections within relatively short periods. In a clinical study by Prabowo et al. [83], sensitivity and specificity for diagnosing the dengue nonstructural protein 1 (NS1) antigen were established for the DEN-NS1-PAD [A diagnostic kit for dengue NS1 antigen testing based on a wax-printed microfluidic paper-based analytical device (μ Pad)]. The authors developed a simple wax-printed paper-based system of the DEN-NS1-PAD that allows specific binding between the reporter antibodies labeled with AuNPs and the dengue NS1 protein in a sample. A visible colorimetric signal is produced at the test line as a result of this binding. This DEN-NS1-PAD has a sensitivity of 88.89 % and a specificity of 86.67 %, indicating its potential to be a valuable diagnostic tool for dengue.
In 2023, Lee et al. [75] created a colorimetric lateral flow immunoassay (LFIA) method with exceptional sensitivity for precise detection of SARS-CoV-2. The authors employed a method that immobilizes antibodies on a nitrocellulose membrane using a CBP31-BC linker for enhanced specificity and sensitivity. It demonstrated a 100 % accuracy rate in analyzing clinical samples and can detect SARS-CoV-2 within around 15 min with a detection limit of 5 × 104 copies/mL. Even in locations with limited resources, this technique shows great promise for accurate and quick testing at the point of care, which could help in limiting the COVID-19 pandemic.
Fluorescent LFAs have emerged as a notable breakthrough in diagnostic testing. Compared to colorimetric LFA, they are significantly more sensitive and specific [84]. Fluorescent LFA detects the analyte in a sample by measuring fluorescence produced by fluorescent labelled molecules on the test line [85]. The outcomes are more accurate and trustworthy. Fluorescent LFA has been used in various industries such as environmental monitoring, food safety, and healthcare to quickly and accurately detect specific substances.
Chavelon et al. [86] developed a fluorescent LFA method for noncompetitive detection of small analytes through a sandwich-type format utilizing an aptamer kissing complex strategy. This method utilizes a fluorescent labelled hairpin aptamer as a signaling agent and an RNA hairpin as a capturing agent absorbed onto the test strip. Conformational changes in the aptamer occur due to the binding of the target molecule, which leads to the kissing interaction between the RNA hairpin and the aptamer, producing a fluorescent signal at the test line.
Fan et al. [87] demonstrated a near-infrared (NIR) fluorescence-based LFIA for the detection of 5-hydroxyflunixin residues in milk. The authors found that NIR dye-mAb (anti-5-hydroxyflunixin antibody) demonstrated higher sensitivity and specificity. In contrast to the AuNP-based LFA with 0.82 ng/mL LOD, the NIR dye-based LFA significantly improved the sensitivity and specificity with a low LOD of 0.073 ng/mL. Hence, NIR dye-mAb can be employed as a suitable alternative to AuNP in the creation of a highly sensitive and specific LFA for quick on-site assessment of specific veterinary drugs.
Seo et al. [88] developed a novel fluorescent LFA using fluorophoreencapsulated nanobeads with excellent sensitivity for detecting SARS-CoV-2. As shown in Fig. 6, a novel method of encapsulating a large Stokes shift fluorophore (single benzene) in polystyrene nanobeads was developed to increase the fluorescence intensity and stability of the probe. In comparison to traditional LFAs based on AuNPs, the newly developed LFA sensor demonstrated a LOD for the SARS-CoV-2 spike protein of 1 ng/mL in about 20 min. In comparison to the commercially available rapid diagnostic kits, this encapsulation approach is more photostable, suitable, and biocompatible for fluorescent probes. This LFA sensor was shown to be reliable for on-site sensitive and fast detection of infectious diseases, viruses, and bacteria.
Chemiluminescent LFA are highly efficient diagnostic tools, consistently producing accurate results with high sensitivity and specificity values [89]. These kits specifically utilize chemiluminescence (CL), which is the emission of light as a result of a chemical reaction, to detect the analyte in a sample [90]. Chemiluminescent LFA kits have a notable advantage over traditional colorimetric approaches in terms of their sensitivity to detect even small quantity of target analyte. Notably, the substrates used in chemiluminescent LFA provide a better signal-to-noise ratio, resulting in more accurate and precise results. These kits have been used in both clinical and field settings due to their user-friendly interface and efficient outcomes.
This CL-based LFA has gained a lot of attention because of its ability to sensitively determine a range of different biomarkers. In 2021, Kim et al. [91] developed a portable CL-LFA platform for the detection of cortisol in human serum. The results demonstrated that the new CL-LFA platform is highly effective for quantitatively measuring cortisol, a stress biomarker, over the linear detection range of 0.78–12.5 μg/dL and with a LOD of 0.342 μg/dL. AuNP probes conjugated with antibodies and horseradish peroxidase were used as a detection module in this study, which generates amplified luminescent signals and thus effectively addressing the limitations of lateral flow-type chromatographic immunoassays. This advance thus allows precise in situ measurements. The results demonstrated very high linearity, suggesting that the developed CL-LFA platform can be a reliable and costeffective alternate tool for clinical diagnosis of cortisol in human serum samples.
CL-based LFAs have achieved remarkable sensitivity in the detection of infectious diseases. Jung and his companions [92] developed an extremely sensitive nanoprobe-based CL-LFA to detect the presence of avian influenza virus. Signal amplifiable nanoprobe was created utilizing mesoporous silica nanoparticles with precisely controlled pore sizes. The nanoprobe selectively immobilizes antibodies (binding receptors) and enzymes (signal transducers) onto the CL-LFA sensor based on their sizes to enhance the detection sensitivity. This nanoprobe could effectively trap large biomolecules such as antibodies on its outside surface, whereas smaller biomolecules such as horseradish peroxidase were embedded within the pores in the inside surface. Nanoprobes with this adaptability presented optimal antigen accessibility and signal amplification, resulting in enhanced detection limits. Utilizing this nanoprobe in a CL-LFA kit, the detection limit of the H3N2 nucleoprotein was 5 pM. Sensitivity tests showed that this is 20–100 times more sensitive than commercial AIV rapid test kits. Furthermore, this platform proved effective in the detection of low pathogenicity avian influenza H9N2, H1N1, and high pathogenicity H5N9 viruses in clinical samples.
Magnetic-based LFAs present a significant breakthrough in the field of rapid diagnostic testing. These LFAs endow traditional LFAs with enhanced sensitivity and specificity by utilizing magnetic nanoparticles [93]. A magnetic LFA test captures a target analyte from a sample by utilizing magnetic nanoparticles conjugated with antibodies [94]. Magnetic based LFA utilizes an external magnetic field that precisely concentrates control antibody conjugated magnetic nanoparticles. In this manner, it reduces the background noise and enhances the specificity of the LFA. Due to their high sensitivity and specificity, magnetic LFAs have been used in clinical diagnostics and other fields as well. Additionally, the use of magnetic particles allows for the automation and multiplexed analysis of samples, enhancing the efficiency of LFA in several diagnostic applications [95].
Bai et al. [96] developed a very sensitive LFA for rapid detection of IAV in humans, utilizing magnetic QD nanobeads (MQBs). The developed MQBs were synthesized by attaching superparamagnetic MnFe2O4 nanobead cores to QDs and specific antibodies. This was done to enrich IAV virions from complex biological matrices and bright fluorescent probe sources for their detection on lateral flow strips. This assay illustrated a remarkable LOD value of 22 pfu/mL within 35 min. MQB-based LFIA distinguished influenza A from other respiratory viruses such as influenza B and adenoviruses, highlighting its sensitivity and specificity. Additionally, the detection and quantification of the fluorescent signal by the integration of smart phone-based fluorescence reader is a promising technique in POCT.
For the detection of rotavirus, Fu et al. [94] developed a magnetic-fluorescent nanocluster LFA kit. A highly sensitive detection platform was created by the integration of magnetite nanoparticles with CdSe-CdS core shell QDs, as shown in Fig. 7. These enriched magnetic nanoclusters enhanced the fluorescent intensity and surpassed the traditional commercial LFA kits by achieving a low LOD of 1.0 × 10 TCID/mL.
To address future pandemic related challenges in POCT, significant progress in PCR, bioelectronic devices, and LFA technologies will be required. In critical situations with limited resources, precise and prompt diagnostic technology is imperative. Although LFA has shown potential for diagnosing infectious diseases, existing limits in sensitivity, quantitation, and selectivity must be addressed. Highly specific labels, such as fluorescent or chemiluminescent markers, to facilitate data tracking and remote diagnostics can help overcome the limitations. This combination can improve the sensitivity and selectivity of these labels. Some advances are also needed to integrate bioelectronics technology into wearable devices, allowing continuous and real-time monitoring of health, which is crucial for early identification and control of infectious diseases. PCR technology, while known for its exceptional sensitivity and specificity, should be enhanced to be more compact and rapid. The advancement of multiplexing capabilities in these technologies will allow for the simultaneous identification of many infections or biomarkers in a single test, delivering rapid and complete diagnostic information. With the rapid progress of microfluidic and microfabrication technologies, we expect that the PCR system can be improved without incurring additional costs and made simpler without compromising its performance. Incorporating upcoming isothermal amplification and lens-free imaging into PCR will lead to a substantial reduction in the size of the PCR system. This optimization will make it suitable for a variety of biomedical applications, especially in developing countries. Collectively, these developments will greatly improve the efficiency of POCT, rendering it an essential instrument in the regulation and handling of forthcoming pandemics by guaranteeing rapid, precise, and easily obtainable diagnostic procedures.
This research was supported by a grant (RS-2024-00331900) from the Ministry of Food and Drug Safety, Republic of Korea.
The authors declare no conflicts of interest.