Rapid, field-deployable diagnostics are critical for detecting high-priority biothreats, yet most platforms lack sensitivity, multiplexing, or usability in resource-limited settings. We present VeriFAST, a compact vertical flow immunoassay (VFI) system integrating automated fluid handling, a nitrocellulose-based multiplex membrane, and smartphone-enabled image processing for real-time analysis. Using a gold nanoparticle sandwich format, VeriFAST simultaneously detects four Tier 1 bacterial antigens LcrV and F1 (Yersinia pestis), FtLPS (Francisella tularensis), CPS (Burkholderia pseudomallei). The system achieved limits of detection of 0.05 ng/mL for F1, 0.0125 ng/mL for CPS and 0.625 ng/mL for LcrV in serum, urine, and soil extracts, with total assay time under 30 min. Reagent stability testing showed that buffers and detection antibodies retained full function for 12 months under ambient or refrigerated storage, whereas capture antibodies began to show signal drift after 3 months at room temperature, even with desiccant protection. Matrix-specific optimization further enabled high reproducibility and low background across complex samples. By combining multiplexing, automated processing, and mobile analytics in a portable format, VeriFAST addresses critical gaps in field diagnostics and offers a scalable solution for biothreat detection in public health, military, and environmental response.

Colorimetric lateral flow immunoassays (LFIA) have revolutionized point-of-care testing (POCT) methods by providing simple, rapid, accessible, and instrumentation-free detection of infectious diseases. However, conventional LFIAs relying on gold nanospheres (GNPs) can suffer from limited sensitivity due to weak colorimetric signal intensity at the test line, restricting their diagnostic potential. To overcome this limitation, we developed a magneto-plasmonics enhanced colorimetric LFIA (mpLFIA)- a breakthrough platform that integrates a novel hybrid nanoparticle system-magnetic gold nanostars (mpGNS). By leveraging dual enhancement mechanisms - magnetic preconcentration and plasmonic amplification through the gold nanostar shell coating of the magnetic core- the mpLFIA achieves unprecedented signal intensity and detection performance. Among the tested mpGNS variants, mpGNS-3- distinguished by its longest spikes and highest branch density- emerged as the most effective colorimetric signal amplifier when evaluated using Rift Valley fever virus (RVFV) nucleoprotein as a model analyte. Our mpLFIA platform achieves an outstanding limit of detection (LOD) of 2.24 pg/mL for RVFV nucleoprotein in 1X PBS buffer, demonstrating a 1000-fold enhancement over conventional GNP-based assays. Our prototype mpLFIA platform, utilizing highly spiked mpGNS-3, exhibits great potential as a powerful bioanalytical tool, combining high sensitivity with practical portability for point-of-care applications.

Rapid biodosimetry at the point of care is critical for efficient triage and clinical management during mass-casualty radiological events. Here, we introduce a power-free biodosimetry platform integrating one-step reverse transcription recombinase polymerase amplification (RT-RPA) with vertical flow immunoassay (VFI) for the multiplex detection of two radiation-responsive transcripts (CDKN1A and DDB2) and housekeeping gene (MRPS5) from fingerstick blood. One-step RT-RPA showed optimal efficiency at 36 °C for 20 min, comparable to two-step RT-RPA and standard PCR. This temperature could be provided by the human body, where the axilla delivered a stable heat source (36.2 °C) with minimal interindividual variability across a range of environmental conditions (from 4 to 42 °C), supporting amplification comparable to thermocycler-based reactions. Amplified products were detected using a capillary-driven VFI platform. The assay demonstrated strong specificity, uniform signal distribution, and reproducibility with intra- and interassay coefficient of variation below 10% and 15%, respectively, across all three transcripts. When applied to ex vivo X-ray irradiated blood samples (1 and 2 Gy), a dose-dependent increase in CDKN1A and DDB2 signals was observed. Receiver operating characteristic analysis demonstrated that discrimination between sham- and 1 Gy-irradiated was not significant, whereas the assay distinguished sham- from 2 Gy-irradiated with area under the curve values of 0.82 (95% CI = [0.66-0.98]; p = 0.0052) and 0.89 (95% CI = [0.76-1.00]; p = 0.0006) for CDKN1A and DDB2, yielding sensitivity/specificity of 84.62%/69.23% and 92.31%/84.62%, respectively. These findings supported the feasibility of the body-heat one-step RT-RPA VFI as a rapid, equipment-free, field-deployable triage tool for large-scale radiation biodosimetry.

The lateral flow immunoassay (LFIA) has become a widely accepted point-of-care diagnostic tool (POCT) due to its simplicity, portability, cost-effectiveness, and rapid biomarker detection capabilities. However, its sensitivity in detecting target analytes has been limited by the visual signals produced by traditional gold nanoparticles. In this study, we introduce a highly sensitive near infrared (NIR) photothermal platform using gold nanostars (GNS) with a tunable plasmon resonance band spanning wavelengths from 700 to 850 nm. The GNS, particularly the GNS-3 probe with its large number of branches, exhibited exceptional light-to-heat conversion efficiency, significantly enhancing photothermal conversion. Using GNS-3 as an efficient photothermal probe, we successfully detected the high-risk pathogen Francisella Tularensis biomarker lipopolysaccharide (FtLPS) as the model analyte, achieving an outstanding limit of detection (LOD) of 3.5 pg/mL for photothermal LFIA. This photothermal LFIA enhances the detection sensitivity nearly 1000-fold compared to traditional colorimetric gold nanosphere-based LFIA. Furthermore, we demonstrate the potential of the photothermal LFIA platform for real-world applicability by detecting ultra-low levels of FtLPS spiked in blood serum samples, achieving an LOD as low as 4 pg/mL. This photothermal LFIA platform shows promise for establishing high-performance photothermal sensing in point-of-care settings and holds great potential for future advancements in rapid, on-site screening of infectious diseases.

The colorimetric lateral flow immunoassay (cLFIA) has gained widespread attention as a point-of-care testing (POCT) technique due to its low cost, short analysis time, portability, and capability of being performed by unskilled operators with minimal requirement of reagents. However, the low analytical sensitivity of conventional LFIA based on colloidal gold nanospheres limits their applications for sensitive detection of trace amounts of target analytes. In this study, we introduced a novel plasmonic-enhanced colorimetric LFIA (PE-cLFIA) platform featuring bimetallic silver-coated gold nanostars (BGNS) with exceptional optical properties, leading to ultrahigh visual color brightness. The BGNS-based PE-cLFIA was successfully applied to detect a model analyte, low-calcium response V (LcrV), a virulence protein factor found in Yersinia pestis, the causative agent of bubonic plague. The PE-cLFIA sensing using BGNS-3 composed of 45 nm silver thickness showed a high visual colorimetric sensitivity with a detection limit as low as 13.7 pg/mL, which was around 50 times more sensitive than that of a traditional gold nanoparticle-based LFIA. In addition, the antibody-conjugated BGNS-3 showed excellent stability over 6 months. To illustrate the potential for clinical applications, we demonstrated that our LFIA platform for detecting LcrV spiked in human serum without any sample preprocessing exhibited a detection limit of 22.8 pg/mL. These results open up new opportunities for developing hybrid nanoparticle systems for sensitive POCT PE-cLFIA screening for infectious disease detection.

Over the past few decades, colorimetric paper-based lateral flow immunoassay (LFIA) has emerged as a versatile analytical tool for rapid point-of-care detection of infectious diseases with high simplicity and flexibility. The LFIA sensitivity is based on color visualization of the antibody-labeled nanoparticles bound with the target analytes at the test line. Therefore, the nanoparticle design is crucial for LFIA sensitivity. The traditional LFIA is based on spherical gold nanoparticles, which usually suffer from poor sensitivity because of very low optical contrast at the test line. To improve the LFIA sensitivity, we have developed an LFIA based on gold nanostars (GNSs) with different branch lengths and sharpness (GNS-1, GNS-2, and GNS-3), which possess higher optical contrast than conventional gold nanospheres (GNSPs). We have selected the bacterium Yersinia pestis as a model analyte system. The effective affinity of GNSPs and GNSs with the Y. pestis fraction 1 (F1) protein was quantitively investigated by colorimetric and optical density measurements of the test line. The results show that GNS-3, which has maximum spike length and branch sharpness, exhibits the highest analytical sensitivity based on the limit of detection of the LFIA readout compared to other GNSs and GNSPs. The detection limit of the Y. pestis F1 antigen was achieved up to 0.1 ng/mL for GNS-3, which is 100 times lower than that for the GNSP at a 1 pmol/L concentration and 10 times lower than that for the reported procedure based on traditional gold nanoparticles. Overall, our prototype LFIA platform based on a highly spiked GNS (GNS-3) exhibits high analytical sensitivity, indicating it to be a promising candidate for routine LFIA application to detect infectious diseases.

This paper presents simple, fast, and sensitive detection of multiple biothreat agents by paper-based vertical flow colorimetric sandwich immunoassay for detection of Yersinia pestis (LcrV and F1) and Francisella tularensis (lipopolysaccharide; LPS) antigens using a vertical flow immunoassay (VFI) prototype with portable syringe pump and a new membrane holder. The capture antibody (cAb) printing onto nitrocellulose membrane and gold-labelled detection antibody (dAb) were optimized to enhance the assay sensitivity and specificity. Even though the paper pore size was relaxed from previous 0.1 μm to the current 0.45 μm for serum samples, detection limits as low as 0.050 ng/mL for LcrV and F1, and 0.100 ng/mL for FtLPS have been achieved in buffer and similarly in diluted serum (with LcrV and F1 LODs remained the same and LPS LOD reduced to 0.250 ng/mL). These were 40, 80, and 50X (20X for LPS in serum) better than those from lateral flow configuration. Furthermore, the comparison of multiplex format demonstrated low cross-reactivity and equal sensitivity to that of the singleplex assay. The optimized VFI platform thus provides a portable and rapid on-site monitoring system for multiplex biothreat detection with the potential for high sensitivity, specificity, reproducibility, and multiplexing capability, supporting its utility in remote and resource-limited settings.

Antibody microarrays have proven useful in immunoassay-based point-of-care diagnostics for infectious diseases. Noncontact piezoelectric inkjet printing has advantages to print antibody microarrays on nitrocellulose substrates for this application due to its compatibility with sensitive solutions and substrates, simple droplet control, and potential for high-capacity printing. However, there remain real-world challenges in printing such microarrays, which motivated this study. The effects of three concentrations of capture antibody (cAb) reagents and nozzle hydrostatic pressures were chosen to investigate three responses: the number of printed membrane disks, dispensing performance, and microarray quality. Printing conditions were found to be most ideal with 5 mg/mL cAb and a nozzle hydrostatic pressure near zero, which produced 130 membrane disks in a single print versus the 10 membrane disks per print before optimization. These results serve to inform efficient printing of antibody microarrays on nitrocellulose membranes for rapid immunoassay-based detection of infectious diseases and beyond.

Yersinia pestis is a Gram-negative bacterium that is the causative agent of plague and is widely recognized as a potential biological weapon. Due to the high fatality rate of plague when diagnosis is delayed, the development of rapid, sensitive, specific, and cost-effective methods is needed for its diagnosis. The Y. pestis low calcium response V (LcrV) protein has been identified as a potential microbial biomarker for the diagnosis of plague. In this paper, we present a highly sensitive, paper-based, vertical flow immunoassay (VFI) prototype for the detection of LcrV and the diagnosis of plague. An antigen-capture assay using monoclonal antibodies is employed to capture and detect the LcrV protein, using a colorimetric approach. In addition, the effect of miniaturizing the VFI device is explored based on two different sizes of VFI platforms, denoted as "large VFI" and "mini VFI." Also, a comparative analysis is performed between the VFI platform and a lateral flow immunoassay (LFI) platform to exhibit the improved assay sensitivity suitable for point-of-care (POC) diagnostics. The analytical sensitivity or limit of detection (LOD) in the mini VFI is approximately 0.025 ng/mL, that is, 10 times better than that of the large VFI platform or 80 times over a standard lateral flow configuration. The low LOD of the LcrV VFI appears to be highly suitable for testing clinical samples and potentially diagnosing plague at earlier time points. In addition, optimization of the gold nanoparticle (AuNP) concentration, nanomaterial plasmonic properties, and flow velocity analysis could improve the performance of the VFI. Furthermore, we developed automated image analysis software that shows potential for integrating the diagnostic system into a smartphone. These methods and findings demonstrate that the VFI platform is a highly sensitive device for detecting the LcrV and potentially many other biomarkers.

Isotachophoresis (ITP) for Pacific Blue (PB) dye using a polydimethylsiloxane (PDMS) microfluidic chip is developed and characterized by determining the types and concentrations of electrolytes, the ITP duration, and the electric field density. Among candidate buffers for the trailing electrolyte (TE) and leading electrolyte (LE), 40 mM borate buffer (pH 9) and 200 mM trisaminomethane hydrochloride (Tris-HCl) (pH 8) were selected to obtain the maximum preconcentration and resolution of the PB bands, respectively. With the selected TE and LE buffers, further optimization was performed to determine the electric field (EF) density and the ITP duration. These ITP parameters showed a 20-170,000 preconcentration ratio from initial PB concentrations of 10 nM-100 fM. Further demonstration was implemented to preconcentrate PB-conjugated lactate dehydrogenase (LDH) using the PDMS microfluidic chip. By utilizing the quenching nature of PB-LDH conjugation, we were able to identify concentrations of LDH as low as 10 ng/mL. This simple PDMS microfluidic chip-based ITP for PB preconcentration enables highly sensitive biological and chemical analyses by coupling with various downstream detection systems.

This study presents a simple, fast, and sensitive method for multiplexing heavy metal detection. Using a chemically patterned microfluidic paper-based analytical device (C-PAD), silane compounds terminating in amine (NH2), carboxyl (COOH), and thiol (SH) were immobilized on a patterned chromatography paper via condensation reactions. Three chromogenic reagents that react distinctly with Ni(II), Cr(VI), and Hg(II) were then covalently coupled to these functional groups. These functionalized C-mu PADs were then evaluated for heavy metals ions detection with spiked water samples. Using a single-plex platform, we achieved detection limits as low as 0.24 ppm for Ni(II), 0.18 ppm for Cr(VI) and 0.19 ppm for Hg(II). The detection limits for this single-plex platform were twice as good as those obtained in previous studies using colorimetric analysis due to the formation of homogeneous metal complexes from immobilized chromogenic reagents. Furthermore, by minimizing cross-reaction with chelating agents and maximizing reaction uniformity, multiplex heavy metal ion detection was successfully demonstrated for lake-water samples spiked with various concentrations of heavy metal ions with high precision. This functionalized C- PAD provides a highly portable and rapid on-site monitoring platform for multiple heavy metal ions detection with extremely high repeatability, which is useful for resource limited areas and developing countries.

A chemically patterned microfluidic paper-based analytical device (C-µPAD) is developed to create fluidic networks by forming hydrophobic barriers using chemical vapor deposition (CVD) of trichlorosilane (TCS) on a chromatography paper. By controlling temperature, pattern size, and CVD duration, optimal conditions were determined by characterizing hydrophobicity, spreading patterns, and flow behavior on various sized fluidic patterns. With these optimal conditions, we demonstrated glucose assay, immunoassay, and heavy metal detection on well-spot C-µPAD and lateral flow C-µPAD. For these assays, standard curves showing correlation between target concentration and gray intensity were obtained to determine a limit of detection (LOD) of each assay. For the glucose assays on both well-spot C-µPAD and lateral flow C-µPAD, we achieved LOD of 13 mg/dL, which is equivalent to that of a commercial glucose sensor. Similar results were obtained from tumor necrosis factor alpha (TNFα) detection with 3 ng/mL of LOD. For Ni detection, a colorimetric agent was immobilized to obtain a stationary and uniform reaction by using thermal condensation coupling method. During the immobilization, we successfully functionalized amine for coupling the colorimetric agent on the C-µPAD and detected as low as 150 μg/L of Ni. These C-µPADs enable simple, rapid, and cost-effective bioassays and environmental monitoring, which provide practically relevant LODs with high expandability and adaptability.

The ammonia odor from the exhaled breath of renal patients is associated with high levels of blood urea nitrogen. Typically, in the liver, ammonia and ammonium ions are converted into urea through the urea cycle. In the case of renal dysfunction, urea is unable to be removed and that causes a buildup of excessive ammonia. As small molecules, ammonia and ammonium ions can be forced into the blood–lung barrier and occur in exhaled breath. Therefore, people with renal failure have an ammonia (fishy) odor in their exhaled breath. Thus, exhaled breath ammonia can be a potential biomarker for monitoring renal diseases during hemodialyis. In this review, we have summarized the source of ammonia in the breath of end-stage renal disease patient, cause of renal disorders, exhaled breath condensate, and breath sampling. Further, various biosensor approaches to detect exhaled ammonia from renal patients and other ammonia systems are also discussed. We conclude with future perspectives, namely colorimetric-based real-time breathing diagnosis of renal failure, which might be useful for prospective studies.

Smartphones and related accessories are rapidly expanding their applications in various fields. Herein we developed a smartphone coupled handheld array reader with the integration of complementary metal oxide (CMOS) image sensor for detecting various toxic gases by colorimetric monitoring approach. In this study, toxic gases such as hydrogen fluoride (HF), chlorine (Cl-2), ammonia (NH3), and formaldehyde (CH2O) were detected using titanium nanoparticles (TiO2 NPs) blended poly (vinyl alcohol) (PVA) hydrogel test strips, which were patterned with chemically responsive dyes. The dye colors changed based on acid-base reactions, and the colorimetric reader monitored the array strips and mapped them in the form of chrominance data. The observed signals transferred to a smartphone that displays the detected toxic gases and their exposure levels with the aid of the smartphone app "Toxic Gas Detection". The colorimetric array reader was precisely constructed with an auto-calibration system to minimize potential errors. Various concentration of toxic gases (0.5-10 ppm) were analyzed and achieved the detection limit of 1 ppm for each gas. Also, various analytical performance including sensitivity, stability, selectivity, and reproducibility analysis proved the reliability and accuracy of the detection system. Additionally, the smartphone app can be installed on any type of tablet, laptop, or other portable device. This study proved that smartphone coupled colorimetric array reader is a suitable system for detecting various toxic gases, chemicals and biochemical analysis with real-time monitoring approach. (C) 2017 Elsevier B.V. All rights reserved.

In the present study, we developed a polypropylene well-integrated complementary metal oxide semiconductor (CMOS) platform to perform the loop mediated isothermal amplification (LAMP) technique for real-time DNA amplification and detection simultaneously. An amplification-coupled detection system directly measures the photon number changes based on the generation of magnesium pyrophosphate and color changes. The photon number decreases during the amplification process. The CMOS image sensor observes the photons and converts into digital units with the aid of an analog-to-digital converter (ADC). In addition, UV-spectral studies, optical color intensity detection, pH analysis, and electrophoresis detection were carried out to prove the efficiency of the CMOS sensor based the LAMP system. Moreover, Clostridium perfringens was utilized as proof-of-concept detection for the new system. We anticipate that this CMOS image sensor-based LAMP method will enable the creation of cost-effective, label-free, optical, real-time and portable molecular diagnostic devices.

CMOS sensors are becoming a powerful tool in the biological and chemical field. In this work, we introduce a new approach on quantifying various pH solutions with a CMOS image sensor. The CMOS image sensor based pH measurement produces high-accuracy analysis, making it a truly portable and user friendly system. pH indicator blended hydrogel matrix was fabricated as a thin film to the accurate color development. A distinct color change of red, green and blue (RGB) develops in the hydrogel film by applying various pH solutions (pH 1-14). The semi-quantitative pH evolution was acquired by visual read out. Further, CMOS image sensor absorbs the RGB color intensity of the film and hue value converted into digital numbers with the aid of an analog-to-digital converter (ADC) to determine the pH ranges of solutions. Chromaticity diagram and Euclidean distance represent the RGB color space and differentiation of pH ranges, respectively. This technique is applicable to sense the various toxic chemicals and chemical vapors by situ sensing. Ultimately, the entire approach can be integrated into smartphone and operable with the user friendly manner.

Abstract. Complementary metal oxide semiconductor (CMOS) image sensors have received great attention for their high efficiency in biological applications. The present work describes a CMOS image sensor-based whole blood glucose monitoring system through a point-of-care (POC) approach. A simple poly-ethylene terephthalate (PET) chip was developed to carry out the enzyme kinetic reaction at various concentrations (110–586  mg/dL) of mouse blood glucose. In this technique, assay reagent is immobilized onto amine functionalized silica (AFSiO2) nanoparticles as an electrostatic attraction in order to achieve glucose oxidation on the chip. The assay reagent immobilized AFSiO2 nanoparticles develop a semi-transparent reaction platform, which is technically a suitable chip to analyze by a camera module. The oxidized glucose then produces a green color according to the glucose concentration and is analyzed by the camera module as a photon detection technique; the photon number decreases when the glucose concentration increases. The combination of these components, the CMOS image sensor and enzyme immobilized PET film chip, constitute a compact, accurate, inexpensive, precise, digital, highly sensitive, specific, and optical glucose-sensing approach for POC diagnosis.

CMOS sensors comprise an important tool in bioscientific applications. This review focuses on CMOS sensor-based molecular diagnostics of DNA, protein, and metabolic molecules. Herein, gene sequencing, DNA–DNA hybridization, single nucleotide polymorphisms (SNP), protein interactions, peptide interactions, antigen–antibody (Ag–Ab) interactions, as well as glucose and cholesterol monitoring using CMOS sensors are discussed along with existing experimental outcomes. CMOS sensor based electrochemical, optical, impedance, dual, continuous, and label-free analysis and their related integration techniques are explained. Moreover, we describe the utilization of a CMOS chip in microarray fabrication, assay platform development, and transducer incorporation for molecular diagnostics. Furthermore, CMOS sensor-based point-of-care (POC) applications, other biological analyses, and the role of nanoparticles in biomolecular sensing are discussed. Future directions include information about the novel integration of CMOS sensor-based molecular diagnostic devices with a central focus towards enhancement of POC approaches. This review is helpful in creating highly sensitive, cheaper, and user-friendly biomedical devices with modern dimensions.

Complementary metal oxide semiconductor (CMOS) image sensors are received great attention for their high efficiency in biological applications. The present work describes a CMOS image sensor-based whole blood glucose monitoring system through a point-of-care (POC) approach. A simple poly-ethylene terephthalate (PET) film chip was developed to carry out the enzyme kinetic reaction at various concentrations of blood glucose. In this technique, assay reagent was adsorbed onto amine functionalized silica (AFSiO2) nanoparticles in order to achieve glucose oxidation on the PET film chip. The AFSiO2 nanoparticles can immobilize the assay reagent with an electrostatic attraction and eased to develop the opaque platform which was technically suitable chip to analyze by the camera module. The oxidized glucose then produces a green color according to the glucose concentration and is analyzed by the camera module as a photon detection technique. The photon number decreases with increasing glucose concentration. The simple sensing approach, utilizing enzyme immobilized AFSiO2 nanoparticle chip and assay detection method was developed for quantitative glucose measurement.