Dual Colorimetric/Electrochemical Detection of Salmonella typhimurium Using a Laser-Induced Graphene Integrated Lateral Flow Immunoassay Strip.

Foodborne illnesses caused by the ingestion of contaminated foods or beverages are a serious concern due to the millions of reported cases per year. It is essential to develop sensitive and rapid detection methods of foodborne pathogens to ensure food safety for producers and consumers. Unfortunately, current detection techniques still suffer from time-consuming operations and the need for highly skilled personnel. Here, we introduce a highly sensitive dual colorimetric/electrochemical detection approach for Salmonella enterica serovar typhimurium (S. typhimurium) based on a laser-induced graphene-integrated lateral flow immunoassay (LIG-LFIA) strip. The LIG electrode was fabricated by laser engraving on a polyimide tape containing a pseudo silver/silver chloride reference electrode from silver sintering and chlorination. Using double-sided tape inserted into the strip, automatic sequential reagent delivery was enabled for the dual-mode signal readout by single-sample loading. A gold-deposited gold nanoparticle strategy was first employed to simultaneously obtain a colorimetric signal for early screening and a signal turn-on electrochemical response for high-sensitivity and -quantitative analysis. A superior performance of the strip was established, characterized by a short analysis time (12 min assay +15 min sample preparation), a broad working concentration range (1 cfu/10 mL to 108 cfu/mL), and the lowest limit of detection (1 ± 0.5 cfu/10 mL; mean ± standard deviation, n = 3) among reported multimode S. typhimurium detection schemes. The strip was successfully applied in the analysis of various food products without any bacterial enrichment or amplification required, and the results were comparable to those of the standard culture method.

Gold nanoparticle core-europium(iii) chelate fluorophore-doped silica shell hybrid nanocomposites for the lateral flow immunoassay of human thyroid stimulating hormone with a dual signal readout.

Hybrid nanocomposite particles composed of a gold core coated with a europium(iii)-chelate fluorophore-doped silica shell (AuNPs@SiO2-Eu3+) have been synthesized and applied as antibody labels in lateral flow immunoassay (LFIA) devices for the determination of human thyroid stimulating hormone (hTSH). Labeling of monoclonal anti-hTSH antibodies with AuNPs@SiO2-Eu3+ nanocomposites allows for both colorimetric and fluorometric observation of assay results on LFIA devices, relying on visible light absorption due to the localized surface plasmon resonance of the Au-core and the fluorescence emission of the Eu(iii)-chelate-modified shell under UV hand lamp irradiation (365 nm), respectively. The possibility for a dual signal readout provides an attractive alternative for LFIAs: instantaneous naked eye observation of the AuNP colorimetric signal as in conventional LFIAs for hypothyroidism detection, and more sensitive fluorescence detection to assess hyperthyroidism. The limits of detection (LOD) for naked eye observation of LFIA devices are 5 µIU mL-1 and 0.1 µIU mL-1 for the colorimetric and fluorimetric detection, respectively. Using the fluorescence detection scheme in combination with a smartphone and digital color analysis, a quantitative linear relationship between the red intensity and the logarithmic concentration of hTSH was observed (R2 = 0.988) with an LOD of 0.02 µIU mL-1. Finally, LFIA devices were effectively applied for detecting hTSH in spiked diluted human serum with recovery values between 100-116%.

Portable smartphone integrated 3D-Printed electrochemical sensor for nonenzymatic determination of creatinine in human urine.

3D printing technologies are an attractive for fabricating electrochemical sensors due to their ease of operation, freedom of design, fast prototyping, low waste, and low cost. We report the fabrication of a simple 3D-printed electrochemical sensing device for non-enzymatic detection of creatinine, an important indicator of renal function. To create the 3D-printed electrodes (3DE), carbon black/polylactic acid (CB/PLA) composite filament was used. The 3DE was activated using 0.5 M NaOH via amperometry prior to use to improve electrochemical performance. To give selectivity for creatinine, the activated 3DE was modified with a copper oxide nanoparticle-ionic liquid/reduced graphene oxide (CuO-IL/rGO) composite. The modified 3DE was characterized using microscopy and electrochemistry. Cyclic voltammetry and amperometry were used to evaluate sensor performance. The modified 3DE provided electrocatalytic activity towards creatinine without enzymes. Under optimal conditions, the modified 3DE directly coupled with a portable smartphone potentiostat exhibited the linear detection range of 0.5-35.0 mM, and the limit of detection was 37.3 µM, which is sufficient for detecting creatinine in human urine samples. Furthermore, the other physiological compounds present in human urine were not detected on the modified 3DE. Therefore, the modified 3DE could be a tool for effective creatinine screening in the urine.

Label free electrochemical DNA biosensor for COVID-19 diagnosis.

The COVID-19 pandemic has significantly increased the development of the development of point-of-care (POC) diagnostic tools because they can serve as useful tools for detecting and controlling spread of the disease. Most current methods require sophisticated laboratory instruments and specialists to provide reliable, cost-effective, specific, and sensitive POC testing for COVID-19 diagnosis. Here, a smartphone-assisted Sensit Smart potentiostat (PalmSens) was integrated with a paper-based electrochemical sensor to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A disposable paper-based device was fabricated, and the working electrode directly modified with a pyrrolidinyl peptide nucleic acid (acpcPNA) as the biological recognition element to capture the target complementary DNA (cDNA). In the presence of the target cDNA, hybridization with acpcPNA probe blocks the redox conversion of a redox reporter, leading to a decrease in electrochemical response correlating to SARS-CoV-2 concentration. Under optimal conditions, a linear range from 0.1 to 200 nM and a detection limit of 1.0 pM were obtained. The PNA-based electrochemical paper-based analytical device (PNA-based ePAD) offers high specificity toward SARS-CoV-2 N gene because of the highly selective PNA-DNA binding. The developed sensor was used for amplification-free SARS-CoV-2 detection in 10 nasopharyngeal swab samples (7 SARS-CoV-2 positive and 3 SARS-CoV-2 negative), giving a 100% agreement result with RT-PCR.

Improvement in sensitivity for lateral flow immunoassay of ferritin using novel device design based on gold-enhanced gold nanoparticles.

This work introduces a low-cost adhesive tape combined with a hydroxylamine/polyvinyl alcohol/polyethylene oxide (HA/PVA/PEO) blend film to fabricate novel devices for improving sensitivity of gold nanoparticle (AuNP)-based lateral flow immunoassays (LFIAs) via two platforms: (1) LFIA device with integrated gold enhancement and (2) LFIA device with two independent sample inlets. The detection of ferritin has been used for proof-of-concept. The adhesive tape inserted in the devices assists to separate two solutions independently flowing from two different inlets toward a nitrocellulose membrane. On-device gold enhancement was achieved by the enlargement of AuNPs via the catalytic reaction of KAuCl4 and HA using the HA/PVA/PEO blend film easily prepared via a solution-casting technique, which could delay the flow of HA released from the film for 180s and improve storage stability of the device. Under optimal conditions evaluated by naked eyes, the gold enhancement (LOD = 0.5 ng/mL) and double-sample inlet (LOD = 2 ng/mL) devices exhibited 20-fold and fivefold higher sensitivity respectively than a conventional device, verifying the sensitivity improvement. Furthermore, the proposed device was successfully detected ferritin in human serum samples within 10 min via naked-eye observation, exhibiting rapidity and simplicity of use, and the capability to perform on-site assays.

Microfluidic Paper-Based Device for Medicinal Diagnosis.

BACKGROUND: The demand for point-of-care testing (POCT) devices has rapidly grown since they offer immediate test results with ease of use, makingthem suitable for home self-testing patients and caretakers. However, the POCT development has faced the challenges of increased cost and limited resources. Therefore, the paper substrate as a low-cost material has been employed to develop a cost-effective POCT device, known as "Microfluidic paper-based analytical devices (µPADs)". This device is gaining attention as a promising tool for medicinal diagnostic applications owing to its unique features of simple fabrication, low cost, enabling manipulation flow (capillarydriven flow), the ability to store reagents, and accommodating multistep assay requirements. OBJECTIVE: This review comprehensively examines the fabrication methods and device designs (2D/3D configuration) and their advantages and disadvantages, focusing on updated µPADs applications for motif identification. METHODS: The evolution of paper-based devices, starting from the traditional devices of dipstick and lateral flow assay (LFA) with µPADs, has been described. Patterned structure fabrication of each technique has been compared among the equipment used, benefits, and drawbacks. Microfluidic device designs, including 2D and 3D configurations, have been introduced as well as their modifications. Various designs of µPADs have been integrated with many powerful detection methods such as colorimetry, electrochemistry, fluorescence, chemiluminescence, electrochemiluminescence, and SER-based sensors for medicinal diagnosis applications. CONCLUSION: The µPADs potential to deal with commercialization in terms of the state-of-the-art of µPADs in medicinal diagnosis has been discussed. A great prototype, which is currently in a reallife application breakthrough, has been updated.

A non-enzymatic disposable electrochemical sensor based on surface-modified screen-printed electrode CuO-IL/rGO nanocomposite for a single-step determination of glucose in human urine and electrolyte drinks.

A non-enzymatic disposable electrochemical sensor coupled with an automated sample pretreatment paper-based device was developed to avoid an additional sample preparation step for glucose determination in human urine and electrolyte drinks. The automated sample pretreatment paper-based device was successfully fabricated by the simple coating of a strong alkaline solution on a patterned wax paper, and then attached on an electrochemical sensor. The nanocomposite of copper oxide nanoparticles, ionic liquid and reduced graphene oxide (CuO-IL/rGO) modified on the screen-printed carbon electrode (SPCE) was created and used as a non-enzymatic electrochemical glucose sensor. The presence of the CuO-IL/rGO nanocomposite on the screen-printed electrode surface was confirmed by transmission electron microscopy (TEM), scanning electron microscopy (SEM) with energy dispersive X-ray spectroscopy (EDX), and X-ray diffraction spectroscopy (XRD). Under optimal conditions, glucose was measured by dropping 100 µL sample solution on the device and detected via chronoamperometry (CA) using a smartphone potentiostat controlled by Android app., providing a rapid current response within 20 s and linearity in a range of 0.03-7.0 mM with a limit of detection (LOD) of 0.14 µM. Furthermore, this developed device was successfully applied for determining glucose levels in human urine and electrolyte drinks, exporting satisfying results correlated with a commercial enzymatic glucose biosensor and labeled values of the commercial products. Therefore, this device could be an alternative device for a non-enzymatic glucose sensor with single-step sample loading, allowing for real-time analysis, low cost, portability, disposability, and on-field measurement.

Novel ractopamine-protein carrier conjugation and its application to the lateral flow strip test for ractopamine detection in animal feed.

In this work, a novel conjugate of ractopamine and bovine serum albumin (RAC-BSA) has been developed via the Mannich reaction, with a mole coupling ratio for RAC-BSA of 9:1. The proposed conjugation method provides a simple and one-step method with the use of fewer reagents compared with other conjugation methods for competitive immunoassays. RAC-BSA conjugation was used to fabricate a competitive lateral flow strip test for RAC detection in animal feed. For sample preparation, RAC was spiked in swine feed purchased from the local markets in Thailand, and methanol and running buffer at a volume ratio of 10:90 was used as extraction buffer. The procedures for sample preparation were completed within 25 min. Under optimal conditions, the limit of detection (LOD), assessed by the naked eye within 5 min, was found to be 1 ng/g. A semi-quantitative analysis was also conducted using a smart phone and computer software, with a linearity of 0.075-0.750 ng/g, calculated LOD of 0.10 ng/g, calculated limit of quantitation of 0.33 ng/g, and good correlation of 0.992. The recoveries were found in the range of 96.4%-103.7% with a relative standard deviation of 2.5%-3.6% for intra- and inter-assays. Comparison of the results obtained by the strip test with those obtained by enzyme-linked immunosorbent assay had a good agreement in terms of accuracy. Furthermore, this strip test exhibited highly specific RAC detection without cross reactivity with related compounds. Therefore, the RAC-BSA conjugation via the Mannich reaction can be accepted as a one-step and easy conjugation method and applied to the competitive lateral flow strip test.

Wax-Assisted One-Step Enzyme-Linked Immunosorbent Assay on Lateral Flow Test Devices.

Lateral flow tests (LFTs) are widely used analytical tools characterized by portability, operator simplicity and short analysis times. A remaining challenge is their limited analytical sensitivity, which in classical immunoassay formats is overcome by enzyme-linked immunosorbent assay (ELISA) formats. The implementation of ELISA to an LFT format however, is hampered by the complexity of the procedure requiring the enzyme substrate addition after sample addition. In this work, a simple method for automation of this procedure without user interference is presented. Originally used sample pads of LFTs have been replaced by hydrophobic wax-modified filter paper-based sample pads to realize a delayed flow a pre-deposited colorimetric ELISA substrate without other alterations to the classical lateral-flow immunoassay format. The performance of the system has been characterized by visualizing flow behavior and final proof-of-concept is provided by a model mouse IgG assay, achieving a limit of detection of 15.8 ng mL-1 from just a single application of the sample solution.

Development of an automated wax-printed paper-based lateral flow device for alpha-fetoprotein enzyme-linked immunosorbent assay.

In this study, a novel wax-printed paper-based lateral flow device has been developed as an alternative approach for an automated and one-step enzyme-linked immunosorbent assay (ELISA). The design pattern consisted of a non-delayed channel, a wax-delayed channel, a test zone and a control zone. This system was easily fabricated on a nitrocellulose membrane using a wax-printing method and then baked in an oven at 100°C for 1min. The four barriers of the wax-delayed channel could delay the flow time for 11s compared to the flow time of the non-delayed channel. To use the device under optimal conditions, alpha-fetoprotein (AFP) was detected at a limit of detection of 1ngmL-1 and assessed with the naked eye within 10min. A colorimetric intensity was also measured using a smart phone and computer software at a linear range of 0.1-100ngmL-1 with a good correlation. Furthermore, the proposed device was successfully applied to detect AFP in human serum. Therefore, the wax-printing demonstrates a user-friendly, easy and quick method for the fabrication of the device, which could be used as a one-step, portable, disposable, low-cost, simple, instrument-free and point-of-care device for the automated ELISA.

Development of a one-step immunochromatographic strip test using gold nanoparticles for the rapid detection of Salmonella typhi in human serum.

An immunochromatographic strip test using gold nanoparticles was developed for the rapid detection of Salmonella typhi (S. typhi) in human serum. The strip test based on the principle of sandwich immunoassay by the specific binding of antigens from S. typhi O901 and antibody of S. typhi O901 on a nitrocellulose membrane. Antibody-gold nanoparticle conjugate was used as the label and was coated onto a glass fiber membrane, which was used as a conjugate pad. To create a test and control zone, antibody of S. typhi O901 and an anti-IgG were dotted on the nitrocellulose membrane, respectively. Positive samples were displayed as red dots at the test and control zones of the nitrocellulose membrane, while negative samples resulted in a red dot only in the control zone. The limit of detection (LOD) was found to be 1.14×10(5) cfu mL(-1), which could be visually detected by the naked eye within 15 min. This strip test provided a lower detection limit and analysis time than a dot blot immunoassay (8.88×10(6) cfu mL(-1) for LOD and 110 min for reaction time). In addition, our immunochromatographic strip test was employed to detect S. typhi in human serum effectively, with high accuracy. This strip test offers great promise for a rapid, simple and low-cost analysis of S. typhi.