Point-of-care Testing Applications Research Articles

Enzymatic reaction modulated gold nanoparticle aggregation-induced photothermal and smartphone readable colorimetry dual-mode biosensing platform for trypsin detection in clinical samples

There is an urgent demand to develop a portable and user-friendly biosensing platform for accurately and rapidly detecting biomarkers in clinical diagnosis. In this work, a photothermal and smartphone-based colorimetric dual-mode biosensing platform was proposed for trypsin assay based on the localized surface plasmon resonance (LSPR) effect of gold nanoparticles (Au NPs). Two competing processes were presented between the protection and the destabilization of dispersed Au NPs, respectively. Bovine serum albumin (BSA) can restrain the Au NPs aggregation due to the surface protection of Au NPs in the salt solution. In the presence of trypsin, the Au NPs are aggregated due to the enzymatic digestion of surface BSA ligand and electrostatic interaction, resulting strong photothermal effect and obvious RGB (Red/Green/Blue) value change (color from red to blue). The whole process can be performed within 1 h and the point-of-care testing (POCT) was realized by using a thermometer and smartphone as signal recorders. The limits of detection (LODs) were calculated to be 6 µg mL−1 by photothermal sensing and 1.2 µg mL−1 by RGB recognition. This biosensing platform was successfully used for trypsin assay in clinical acute pancreatitis samples and showed great consistency with clinical diagnostic results, validating the potential POCT application.

Mechanical Timer-Actuated Fluidic Dispensing System: Applications to an Automated Multistep Lateral Flow Immunoassay with High Sensitivity.

In this study, we present a fluidic dispensing system that can automate the sequential fluidic delivery of multiple reagents for lateral flow assays. Highly sensitive assays typically require multiple solution-based sequences, including washing steps and signal amplification. However, implementation of these types of sequences on an automated and highly sensitive point-of-care testing (POCT) platform remains challenging. Our platform consists of two disposable cartridges with reagent chambers and a test strip and an instrument that has a mechanical timer to actuate the cam-follower-gear components. The timer rotation sequentially shifts the position of the chambers and loads the reagents to the test paper strip. The dispensing intervals are controlled at a variation of <1% within a total actuation time of 60 min. Unlike other POCT devices, the timing of fluid delivery in our timer-actuated platform is not dependent on the selection of substrates and reagents, and the unique approach to fluidic delivery results in no reagent overlap or carryover, minimal reagent loss, and highly accurate fluidic timing control for highly sensitive solution-based assays. As a model application, the proposed platform applies a gold enhancement solution to amplify the detection signal and detect prostate-specific antigen with a limit of detection of 86 pg/mL within 27 min. This platform provides an opportunity for solution-based POCT applications with high sensitivity, thereby satisfying the requirement for user-friendly operations in resource-limited settings.

Design of portable electrochemiluminescence sensing systems for point-of-care-testing applications

Point-of-care testing (POCT) technology is highly desirable for clinical diagnosis, healthcare monitoring, food safety inspection, and environment surveillance, because it enables rapid detection anywhere, anytime, and by anyone. Electrochemiluminescence (ECL) has been widely used in chemo-/bio analysis due to its advantages such as high sensitivity, simplicity, rapidity and easy to control, and is now attracting increasing attention for POCT applications. However, to realize the accurate on-site quantitation, it is still challenging to develop portable devices which can precisely collect, analyze, transmit and display the ECL signals. This review will focus on how to develop a portable ECL device by summarizing recent examples and analyzing their key components part by part. Then the possible solutions to the existing challenges in the development and applications of portable ECL devices are summarized and discussed in detail, followed by offering future perspectives. We attempted to provide an appealing viewpoint to inspire interested researchers to comprehend and explore portable ECL sensing systems for practical applications and even commercialization.

An impedometric sensor based on boronic acid @ plastic chip electrode for sensitive detection of prostate cancer biomarker spermine

Elevated levels of the aliphatic biogenic amine spemine in human urine and plasma has been considered as a biomarker for different types of cancers particularly, prostate cancer (PCa). Rapid measurement of spermine in human body fluids with high sensitivity is of great significance for the early clinical diagnosis of PCa. However, accurate estimation of the change in spermine concentration in the order of sub micromolar level is required for point-of-care testing (POCT) drive. Herein, a spermine specific receptor has been successfully attached onto the surface of a home-made gold modified plastic chip electrode (PCE). This electrosensor showed good repeatability and reproducibility in the electrochemical measurement of spermine when tested via impedance spectroscopy over a wide concentration range 10-3-10-11 M. Attachment of the receptor using gold thiol chemistry has been confirmed by SEM-EDX as well as Raman spectroscopy. The biosensor (i.e. boronic acid fabricated PCE) demonstrated good linearity over a wide concentration window (10−3-10−11 M) with R2 = 0.983, 0.9388, and 0.9454 for aqueous, urine, and blood plasma samples, respectively. The interference studies have also been performed with other biogenic polyamines, which revealed remarkable specificity of the electrosensor for spermine. The ultralow limit of detection (10 pM) undoubtedly reveals the potential of the electrochemical biosensing platform to be useful for POCT applications targeted towards the early diagnosis of PCa.

Multiplexed lateral flow assay integrated with orthogonal CRISPR-Cas system for SARS-CoV-2 detection

The development of field-deployable detection platform amenable for multiplexed genes testing will significantly improve the efficiency and reliability during point-of-care testing (POCT) applications. In this regard, an orthogonal CRISPR-Cas-mediated multiplexed lateral flow assay (designated as OC-MLFA) is proposed for SARS-CoV-2 genome detection. Taking the advantage of activation and cleavage preferences between Cas12a and Cas13a, orthogonal (two-independent-channel signal readout) CRISPR-Cas system is investigated. Lateral flow strips with two target lines are designed to accommodate the orthogonal CRISPR system. The interference between Cas12a and Cas13a channels can be effectively eliminated via the elaborate nucleic acids and lateral flow strips design. The high preamplification efficiency from reverse transcription recombinase polymerase amplification (RT-RPA) and Cas enzyme mediated trans-cleavage process bring the sensitivity of our OC-MLFA method to 10 copies per test (30 μL). Nasopharyngeal swab clinical samples with different cycle threshold (Ct) values according to the RT-PCR method were analyzed with the proposed OC-MLFA, during which 76 out of 76 detection accuracy was obtained. Featured with the multiplexed genes detection simultaneously in one reaction and colorimetric readout through single strip, the OC-MLFA we proposed herein ensures great accuracy and efficiency, which endows promising field-deployable POCT application feasibility.

Design and implementation of a low‐cost portable reader for thermometric lateral flow immunoassay

AbstractLateral flow immunoassay (LFIA) is a widely used tool for point‐of‐care testing (POCT). Although the method is fast and inexpensive, it provides only qualitative or semi‐quantitative information, which limits the scope of its applications in POCT. Here, we report the development of a low‐cost, portable LFIA reader based on photothermal detection of reporters (such as colloidal gold and colored latex beads) captured on LFIA strips using a low‐power green laser as the heating source and a single‐element infrared sensor as the detector. For 40‐nm colloidal gold, we measured a detection limit of 3 × 105 particles/mm2 by laser irradiation of the reporters at 532 nm with an intensity of 20 W/cm2. The photothermal detection enables a 10‐fold enhancement in sensitivity over color visualization with the naked eye. More importantly, with the use of the control line intensities as internal calibrants, the results so obtained are quantitative and useful to support critical decision‐making in POCT, as demonstrated by gold‐based assays for human chorionic gonadotropin and human immunodeficiency virus as well as latex‐based assays for nucleocapsid protein of the SARS‐CoV‐2 virus.

Plasmonic Fluorescence Enhancement in Diagnostics for Clinical Tests at Point-of-Care: A Review of Recent Technologies.

Fluorescence-based biosensors have widely been used in the life-sciences and biomedical applications due to their low limit of detection and a diverse selection of fluorophores that enable simultaneous measurements of multiple biomarkers. Recent research effort has been made to implement fluorescent biosensors into the exploding field of point-of-care testing (POCT), which uses cost-effective strategies for rapid and affordable diagnostic testing. However, fluorescence-based assays often suffer from their feeble signal at low analyte concentrations, which often requires sophisticated, costly, and bulky instrumentation to maintain high detection sensitivity. Metal- and metal oxide-based nanostructures offer a simple solution to increase the output signal from fluorescent biosensors due to the generation of high field enhancements close to a metal or metal oxide surface, which has been shown to improve the excitation rate, quantum yield, photostability, and radiation pattern of fluorophores. This article provides an overview of existing biosensors that employ various strategies for fluorescence enhancement via nanostructures and have demonstrated the potential for use as POCT. Biosensors using nanostructures such as planar substrates, freestanding nanoparticles, and metal-dielectric-metal nanocavities are discussed with an emphasis placed on technologies that have shown promise towards POCT applications without the need for centralized laboratories.

Electrochemical paper-based biosensors for point-of-care diagnostics: Detection methods and applications

Personalized health care (PHC) includes personalized, preventive, predictive and participatory approaches that are significant in new diagnostics. This personal health care requires fast, accurate and minimally invasive diagnostic tools that make it possible to evaluate and monitor the process of disease by diagnosing specific disease biomarkers. Point-of-care testing (POCT) involves a wide range of diagnostic tools that meet this purpose. Electrochemical paper-based devices (ePADs) have been introduced as simple, inexpensive, portable and disposable measurement devices to be used in many POCT applications, especially in handling emergencies and outpatient as well as remote usages. Electrochemical detection is a real quantitative detection method with better sensitivity, selectivity and detection limits than indirect measurement methods. In recent years, there has been a revolution in quantitative detections by POCT, thanks to the benefits of electro­chemical sensors and paper substrates. In this paper, recent developments in ePADs, focus­ing on the properties of paper, reasons for its use in the devices, techniques of device and electrode fabrications, and their application particularly in clinical diagnosis, are reviewed.

Rapid One-Tube RPA-CRISPR/Cas12 Detection Platform for Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a severe health threat causing high-level morbidity and mortality in health care environments and in community settings. Though existing diagnostic methods, including PCR and culture-based methods, are routinely used in clinical practice, they are not appropriate for rapid point-of-care testing (POCT). Recently, since the development of the CRISPR/Cas technology, new possibilities for rapid point-of-care detection have emerged. In this study, we developed a rapid, accurate, and contamination-free platform for MRSA detection by integrating recombinase polymerase amplification (RPA) with the Cas12 system into one tube. Using this approach, visual MRSA detection could be achieved in 20 min. Based on the one-tube RPA-CRISPR/Cas12a platform, the assay results are visualized by lateral flow test strips (LFS) and fluorescent-based methods, including real-time and end-point fluorescence. This platform allows specific MRSA detection with a sensitivity of 10 copies for the fluorescence method and a range of 10–100 copies for the LFS. The results of 23 samples from clinical MRSA isolates showed that the coincidence rate was 100% and 95.7% of the fluorescence method and LFS, respectively, compared to qPCR. In conclusion, the one-tube RPA-CRISPR/Cas12a platform is an effective method for MRSA detection with significant potential in future practical POCT applications.

A two-step competition assay for visual, sensitive and quantitative C-reactive protein detection in low-cost microfluidic particle accumulators

Monitoring C-reactive protein (CRP) levels in point-of-care testing (POCT) scenarios is of great value in early diagnosis, prevention and treatment of inflammation in patient’s home, development country as well as other resource-limited settings. Here, we reported a sensitive, economic and simple biosensing approach for visual and sensitive CRP detection by trapping and accumulating microparticles in a photolithography-free, low-cost microfluidic chip. A competition assay among microparticles, magnetic nanoparticles and CRP antigen is utilized to quantify the CRP concentrations. When CRP antigen is present, fewer magnetic nanoparticles can bind to antibody-conjugated microparticles, leaving the free microparticles flowing and trapping in a microfluidic particle accumulation chip after magnetic separation, and forming a length-quantifiable visual bar, which is proportional to the concentrations of CRP in detection samples. Therefore, the target protein can be quantitatively detected by reading the length-quantifiable visual bar in a microfluidic channel by the naked eyes. Of note, a novel two-step competition assay was utilized to achieve the highly sensitive and specific CRP detection with a limit of detection (LOD) of ~ 32 pg/mL, and a wide detection range from 100 pg/mL to 10 µg/mL. The developed method also showed high selectivity, good accuracy and great stability to different temperature and incubation time. More importantly, the design of the sensor could be applicable for detecting any other biomarkers feasibly, thus providing a universal platform for visual, sensitive and economic clinical biomarker measurement by the naked eyes for POCT applications without any auxiliary equipment.

Visible-light and near-infrared fluorescence and surface-enhanced Raman scattering point-of-care sensing and bio-imaging: a review.

This review article deals with the concepts, principles and applications of visible-light and near-infrared (NIR) fluorescence and surface-enhanced Raman scattering (SERS) in in vitro point-of-care testing (POCT) and in vivo bio-imaging. It has discussed how to utilize the biological transparency windows to improve the penetration depth and signal-to-noise ratio, and how to use surface plasmon resonance (SPR) to amplify fluorescence and SERS signals. This article has highlighted some plasmonic fluorescence and SERS probes. It has also reviewed the design strategies of fluorescent and SERS sensors in the detection of metal ions, small molecules, proteins and nucleic acids. Particularly, it has provided perspectives on the integration of fluorescent and SERS sensors into microfluidic chips as lab-on-chips to realize point-of-care testing. It has also discussed the design of active microfluidic devices and non-paper- or paper-based lateral flow assays for in vitro diagnostics. In addition, this article has discussed the strategies to design in vivo NIR fluorescence and SERS bio-imaging platforms for monitoring physiological processes and disease progression in live cells and tissues. Moreover, it has highlighted the applications of POCT and bio-imaging in testing toxins, heavy metals, illicit drugs, cancers, traumatic brain injuries, and infectious diseases such as COVID-19, influenza, HIV and sepsis.

A gas-pressure-assisted ratiometric atomic flame assay for the point-of-care testing of tumor-cell-derived exosomes.

The multicolor-based point-of-care testing (POCT) of tumor cell-derived exosomes is of vital importance for understanding tumor growth and metastasis. Multicolor-based ratiometric signals most often rely on molecular optics, such as fluorescence resonance energy transfer (FRET)-dependent molecular fluorescence and localized surface plasmon resonance (LSPR)-related molecular colorimetry. However, finding acceptable FRET donor-acceptor fluorophore pairs and the kinetically slow color responses during size-related molecular colorimetry have greatly impeded POCT applications. Herein, an atomic flame was used to develop a visual sensing platform for the POCT of tumor-cell-derived exosomes. In comparison with common molecular optics, the atomic flame possessed the advantages of providing both a variety of ratiometric flame signals and fast response sensitivity. The integration of a gas-pressure-assisted flame reaction and dual-aptamer recognition guaranteed the sensitive and selective analysis of exosomes with a low limit of detection (LOD) of 7.6 × 102 particles per mL. Such a novel optical signal will inspire the development of more user-friendly POCT approaches.

Instant Preparation of Ultraclean Gold Nanothorns under Ambient Conditions for SERS Kit-Enabled Mobile Diagnosis.

Availability of surface-enhanced Raman scattering (SERS) substrates with good stability, high sensitivity, and a clean surface is crucial for the practical usefulness of the SERS technology in biochemical sensing, especially for point-of-care testing (POCT). Hereby, we develop a "ready-to-use" SERS kit, which requires only 20 s to fabricate ultraclean gold nanothorn (AuNT)-based SERS chips under ambient conditions with simple solution processing steps. By varying the thickness of the pre-coated platinum (Pt) nanolayer, we can control the size and number density of the grown AuNT. Taking advantage of the ultraclean surface of the instantly obtained fresh AuNT, Raman reporter molecules can also be immediately modified, by means of which specific detection of three analytes including H2O2, NO2-, and ClO- is realized. Furthermore, we propose the concept of an SERS kit and apply it to smartphone-based Raman analysis for POCT applications. This on-site preparation method solves the long-standing challenges hindering the practical use of SERS substrates, such as complicated fabrication processes, interference of residual surfactants, poor surface stability, and easy contamination. Besides performing SERS analysis conveniently and quickly, this SERS kit-enabled POCT technology can integrate remote data terminals and medical resources, which shows great potential for environmental protection or online-healthcare systems.

Hydrogel Paper-Based Analytical Devices: Separation-Free In Situ Assay of Small-Molecule Targets in Whole Blood.

While colorimetric-based assays are very convenient to determine biomarkers in point-of-care testing (POCT), they often suffer from pretreatment procedures for separation of plasma or serum from whole blood samples. Here, we report a simple colorimetric paper-based analytical device (c-PAD) that is capable of performing sample-to-answer analysis by directly dropping the whole blood sample on paper. This is accomplished by utilizing sodium alginate hydrogel, which exhibits a nanometer-scale porous structure to effectively prevent the passage of large red blood cells and hemoglobin molecules, to encapsulate enzymes and chromogenic reagents. As the small targets in the blood sample enter the sensing region to trigger a chromogenic reaction, the resulting color signal is recorded by a smartphone. The interference from the red blood to the color signal can be completely avoided without the requirement of any separation process. The analytical performance of the method is evaluated by assaying glucose in real blood samples. The results show that rapid and accurate analysis can be achieved with the limit of detection as low as 0.12 mM. In addition, simultaneous detection of different targets (glucose, cholesterol, and triglycerides) in whole blood can be achieved by fabricating c-PAD with multiple sensing regions. Owing to its several essential advantages including an extremely simple procedure for fabrication, sample-to-answer analysis without tedious pretreatment, and capability to perform high-throughput analysis, the proposed c-PAD will be of great value in POCT applications of whole blood samples.

After Theranos: Using point-of-care testing to advance measures of health biomarkers in human biology research.

The rise and fall of the health technology startup Theranos is emblematic of the promise and peril of point-of-care testing (POCT). Instruments that deliver immediate results from minimally invasive samples at the location of collection can provide powerful tools to deliver health data in clinical and public health contexts. Yet, POCT availability is driven largely by market interests, which limits the development of inexpensive tests for diverse health conditions that can be used in resource-limited settings. These constraints, combined with complex regulatory hurdles and substantial ethical challenges, have contributed to the underutilization of POCT in human biology research. We evaluate current POCT capabilities and limitations, discuss promising applications for POCT devices in resource-limited settings, and discuss the future of POCT. As evidenced by publication trends, POCT platforms have rapidly advanced in recent years, gaining traction among clinicians and health researchers. We highlight POCT devices of potential interest to population-based researchers and present specific examples of POCT applications in human biology research. Several barriers can limit POCT applications, including cost, lack of regulatory approval for non-clinical use, requirements for expensive equipment, and the dearth of validation in remote field conditions. Despite these issues, we see immense potential for emerging POCT technology capable of analyzing new sample types and used in conjunction with increasingly common technology (e.g., smart phones). We argue that the fallout from Theranos may ultimately provide an opportunity to advance POCT, leading to more ethical data collection and novel opportunities in human biology research.

Optical fiber amplifier and thermometer assisted point-of-care biosensor for detection of cancerous exosomes

Developing a simple, reliable and sensitive point-of-care testing (POCT) biosensor for cancerous exosomes detection is crucial to early cancer diagnosis and prognosis. Herein, a colorimetric and photothermal POCT biosensor was demonstrated based on rolling circle amplification (RCA) mediated 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS)‑H2O2 system for sensitive detection of cancerous exosomes. In the presence of target exosomes, RCA could be triggered and produced multiple units that hybridized to horseradish peroxidase (HRP) modified DNA probes. Colorless ABTS would be converted by HRP into the oxidized form with strong near-infrared absorbance, enabling colorimetric and photothermal detection of exosomes. Then, colorimetric and photothermal responses were recorded by optical fiber amplifier and forehead thermometer, respectively, enabling hand-held detection. Moreover, EpCAM aptamer was used as biorecognition element for exosomes secreted by MCF-7 breast cancer cells so that the point-of-care biosensor exhibited excellent specificity. Furthermore, the point-of-care biosensor could effectively detect the cancerous exosomes in 30% fetal bovine serum with lots of normal exosomes. Moreover, the biosensor could avoid the influence of the external environment, including surrounding light and temperature. These results suggest that the proposed strategy has great potential as a powerful tool for POCT application of exosomes in future diagnosis.

An integrated electrochemical POCT platform for ultrasensitive circRNA detection towards hepatocellular carcinoma diagnosis

Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related death. Circ-CDYL, one of the circular RNAs (circRNAs), is recognized as an independent marker for HCC early diagnosis. Point-of-care testing (POCT) of circRNA is essential and in great demand for clinical applications. Herein, we report a fully integrated electrochemical POCT platform for circRNA detection based on Au nanoflowers (AuNFs)/peptide nucleic acid (PNA) modified carbon-fiber microelectrode (CFME). PNA is applied as the recognition element, highly specified for a back-splice junction of circRNA. AuNFs increased active site for PNA probes, improving target-capturing efficiency at an ultralow level. The platform provides a linear range of 10 fM to 1 μM, with a detection limit as low as 3.29 fM. This biosensor demonstrates high specificity towards one-base mismatch and is stable for up to 24 days. The analytical performance has also been verified in human serum samples, demonstrating the potential utility in clinical POCT applications for HCC.

Soft Electrochemical/Electronic Devices and Their Applications in Point-of-Care Testing

Using soft materials (e.g., paper, hydrogel) to replace the traditional hard materials (e.g., glass, silicon) as substrates have become a recent trend in developing point-of-care testing (POCT) platforms. And through integration of the soft materials with electrochemical or electric devices can further extend the applications of POCT. In our recent work, we used four kinds of soft materials, including paper, PDMS, hydrogel and liquid marble, as the new soft substrates of POCT platforms and realized their new applications in POCT. For example, by integration of the electrodes and hydrophobic channels on paper through simply writing method using home-made pens, the fabricated paper-based electrochemical devices have been successfully applied to detect glucose in artificial urine and melamine in sample solutions. By fabrication of MWNTs/PDMS fibers as multifunctional sensors and integration them on a glove, the measurement of gesture recognition and temperature have been realized by the “smart” glove. Through injection of liquid metal into the 3D helical structure of hydrogel and combination the hydrogel with electronic sensing device, the temperature, UV sensing and EMG signal detections have been performed by the wearable, multifunctional hydrogel device. By using liquid marble as a separator and small device for isoelectric focusing, the POCT separation and analysis of proteins have been realized. The proposed new soft platforms show wide application potential in POCT.

Equipment-free quantitative determination of urea based on paper-based sensor via urease-mediated chitosan viscosity change

In this study, a paper-based sensor combined with visual distance-readout technique for point of-care testing (POCT) of urea was developed by urease-mediated chitosan viscosity change. A series of factors that affect the performance of the sensor were investigated, including the type of filter paper, chitosan concentration, acetic acid concentration and enzymatic reaction conditions. Under optimal conditions, the proposed method for urea determination has good linearity between 3.8–15.1 mM. The limit of quantitation is 3.8 mM. Finally, the paper-based sensor was successfully applied to the determination of urea in two diesel exhaust fluid (DEF) samples. The recoveries of urea were 91.4 % and 109.9 % in DEF-1 and DEF-2, respectively. The present study provides a novel approach, which integrates paper-based sensor and visual distance-readout technique, for monitoring urea in POCT application, especially in remote or resource-limited regions.

Development of an Inkless, Visual Volumetric Chip Operated with a Micropipette

Visual volumetric chips based on gas-generating reactions can potentially be applied to instrument-free point-of-care testing (POCT). However, the structures of conventional chips and their detection methods are typically complicated due to two technical issues: (1) ink, as a distance marker, must be injected into a specified position before sample introduction, and (2) the gas-generating reaction must be initiated in a sealed state. In this study, an inkless volumetric chip operated with a micropipette was developed. The volumetric chip consisted of a reaction well (R well), a substrate well (S well) containing a H2O2 solution as an O2 generator, and a channel connecting the upper side of the R well and the lower side of the S well. Sample injection into the R well is followed by the insertion of a micropipette that is used to exert a negative pressure, which subsequently drives the transfer of a large portion of the H2O2 solution to the R well to initiate the gas-generating reaction. Meanwhile, a small portion of the H2O2 solution remains in the channel for use as a distance marker. The performance of the volumetric chip was evaluated by detecting Salmonella typhimurium, affording a limit of detection of 10 CFU within 20 min for culture samples and within 75 min for spiked milk samples. The volumetric chip developed herein is advantageous for POCT applications due to its simple structure and detection process.