System For Simultaneous Detection Research Articles

Development of a qPCR assay and a LAMP assay for Verticillium longisporum detection and a triplex qPCR assay for simultaneous detection of V. longisporum, Leptosphaeria biglobosa and L. maculans from canola samples

Verticillium wilt, Verticillium stem striping, and Verticillium stripe, are common disease names that all denote infection caused by Verticillium longisporum, on canola, or other Brassica crops. In this study, a quantitative PCR (qPCR) assay and a loop-mediated isothermal amplification (LAMP) assay were developed for the detection of V. longisporum from canola stem samples. Both assays are specific to V. longisporum at the species level and ubiquitous at the strain level across all whole-genome sequenced strains. The low limit for positive detection of the two assays is 1 pg fungal DNA in a 20-µL reaction or 700 fungal cells in 100-mg plant tissue. The qPCR assay was combined with the duplex qPCR assay for the two blackleg pathogens, Leptosphaeria biglobosa and L. maculans to constitute a triplex qPCR system for simultaneous detection of all three pathogens. The usefulness of this triplex qPCR system was verified on canola samples collected from various locations in Alberta, Canada. Using this triplex qPCR system, V. longisporum was detected from one sample, while the two blackleg pathogens were detected at higher frequencies. Since it is sometimes difficult to differentiate Verticillium stripe and blackleg on Alberta canola samples based on visual symptoms, the triplex qPCR system is an important tool for the detection of V. longisporum, especially when its presence is masked or obscured by symptoms of blackleg.

Polythiophene-wrapped Chitosan Nanofibrils with a Bouligand Structure toward Electrochemical Macroscopic Membranes.

Exploring structural biomimicry is a great opportunity to replicate hierarchical frameworks inspired by nature in advanced functional materials for boosting new applications. In this work, we present the biomimetic integration of polythiophene into chitosan nanofibrils in a twisted Bouligand structure to afford free-standing macroscopic composite membranes with electrochemical functionality. By considering the integrity of the Bouligand structure in crab shells, we can produce large, free-standing chitosan nanofibril membranes with iridescent colors and flexible toughness. These unique structured features lead the chitosan membranes to host functional additives to mimic hierarchically layered composites. We used the iridescent chitosan nanofibrils as a photonic platform to investigate the host-guest combination between thiophene and chitosan through oxidative polymerization to fabricate homogeneous polythiophene-wrapped chitosan composites. This biomimetic incorporation fully retains the twisted Bouligand organization of nanofibrils in the polymerized assemblies, thus giving rise to free-standing macroscopic electrochemical membranes. Our further experiments are the modification of the biomimetic polythiophene-wrapped chitosan composites on a glassy carbon electrode to design a three-electrode system for simultaneous electrochemical detection of uric acid, xanthine, hypoxanthine, and caffeine at trace concentrations.

A Custom-Tailored Multichannel Pressure Monitoring System Designed for Experimental Surgical Model of Abdominal Compartment Syndrome.

In experimental medicine, a wide variety of sensory measurements are used. One of these is real-time precision pressure measurement. For comparative studies of the complex pathophysiology and surgical management of abdominal compartment syndrome, a multichannel pressure measurement system is essential. An important aspect is that this multichannel pressure measurement system should be able to monitor the pressure conditions in different tissue layers, and compartments, under different settings. We created a 12-channel positive-negative sensor system for simultaneous detection of pressure conditions in the abdominal cavity, the intestines, and the circulatory system. The same pressure sensor was used with different measurement ranges. In this paper, we describe the device and major experiences, advantages, and disadvantages. The sensory systems are capable of real-time, variable frequency sampling and data collection. It is also important to note that the pressure measurement system should be able to measure pressure with high sensitivity, independently of the filling medium (gas, liquid). The multichannel pressure measurement system we developed was well suited for abdominal compartment syndrome experiments and provided data for optimizing the method of negative pressure wound management. The system is also suitable for direct blood pressure measurement, making it appropriate for use in additional experimental surgical models.

Simultaneous real-time detection of fractional exhaled nitric oxide and end-tidal carbon dioxide by quantum cascade laser absorption spectroscopy

The simultaneous detection of fractional exhaled nitric oxide (FeNO) and end-tidal carbon dioxide (ETCO2) is of great importance for the distinguishing and diagnosis of asthma and chronic obstructive pulmonary disease (COPD), providing more comprehensive information on respiratory disorders. This work demonstrates a simultaneous ETCO2 and FeNO detection system based on quantum cascade laser absorption spectroscopy (QCLAS) technology was presented. The system employs wavelength modulation spectroscopy (WMS) technology and the Herriott multi-pass cell, achieving a detection limit of 2.82 ppb for nitric oxide (NO) and 0.05 % for carbon dioxide (CO2). Real-time exhalation measurements were performed on volunteers with varying ETCO2 and FeNO levels, and the results of the test can accurately distinguish whether the corresponding volunteer was healthy, had asthma or COPD. The effect of exhalation flow rate on the concentration of the two gases was explored. A range of expiratory flow rates were tested in the flow rate interval from 1 to 4 L/min, and there was always an inverse relationship between expiratory flow rate and FeNO concentration, but flow rate changes did not affect ETCO2 concentration. The results indicate that this detection system can simultaneously and effectively measure ETCO2 and FeNO concentrations in real-time.

Near-infrared wide-range dual-gas sensor system for simultaneous detection of methane and carbon monoxide in coal mine environment

To effectively monitor gas explosion and coal spontaneous combustion in coal mine, an intrinsically safe and explosion-proof dual-gas sensor system was developed for methane (CH4) and carbon monoxide (CO) measurement. A distributed feedback (DFB) laser was used to scan two adjacent absorption lines (CH4: 4284.51 cm−1, CO: 4285.01 cm−1). Two measurement ranges of 0–500 parts per million by volume (ppmv) and 0–300,000 ppmv for CH4 and 0–100 ppmv and 0–4000 ppmv for CO were realized using a ∼25 cm single-pass gas cell (SPGC) and a ∼25 m multi-pass gas cell (MPGC), respectively. A gas pre-treatment module was used to remove moisture and aerosols from gas samples. A temperature compensation algorithm was adopted to deal with ambient temperature change. Based on the Allan deviation analysis, the limit of detection (LoD) of CH4 is 0.19 ppmv and the LoD of CO is 0.12 ppmv, respectively, at an averaging time of 0.5 s. A continuous two-day CH4 measurement was performed to demonstrate the long-term stability and environmental monitoring capability. The sensor system was successfully deployed in coal mine gas analysis at Longjiapu, Changchun City, China.

Nucleic Acid Detection through RNA-Guided Protease Activity in Type III-E CRISPR-Cas Systems.

RNA-guided protease activity was recently discovered in the type III-E CRISPR-Cas systems (Craspase), providing a novel platform for engineering a protein probe instead of the commonly used nucleic acid probe in nucleic acid detection assays. Here, by adapting a fluorescence readout technique using the affinity- and fluorescent protein dual-tagged Csx30 protein substrate, we have established an assay monitoring Csx30 cleavage by target ssRNA-activated Craspase. Four Craspase-based nucleic acid detection systems for genes from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), norovirus, and the influenza virus (IFV) were reconstituted with demonstrated specificity. The assay could reliably detect target ssRNAs at concentrations down to 25 pM, which could be further improved approximately 15 000-fold (ca. 2 fM) by incorporating a recombinase polymerase isothermal preamplification step. Importantly, the species-specific substrate cleavage specificity of Craspase enabled multiplexed diagnosis, as demonstrated by the reconstituted composite systems for simultaneous detection of two genes from the same virus (SARS-CoV-2, spike and nsp12) or two types of viruses (SARS-CoV-2 and IFV). The assay could be further expanded by diversifying the fluorescent tags in the substrate and including Craspase systems from various species, thus potentially providing an easily adaptable platform for clinical diagnosis.

Detection and quantification of integrated vector copy number by multiplex droplet digital PCR in dual-transduced CAR T cells

The success of chimeric antigen receptor (CAR) Tcell therapies in refractory hematologic malignancies has prompted investigation of their efficacy in solid tumors. AUTO6NG is a dual-transduced GD2-targeting CAR that encodes distinct modules designed to enhance Tcell activity in relapsed/refractory neuroblastoma. The ability to detect and precisely quantify vector copy number (VCN) for each integrated vector is essential for assessing the effect of each module on Tcell tumor infiltration, persistence, and clinical activity. Droplet digital PCR (ddPCR) enables accurate, sensitive, and absolute quantification of specific nucleic acid sequences. Compared to standard detection of two targets, multiplex ddPCR assays allow simultaneous detection of up to four targets by selective modulation of signal amplitude while retaining the ability to quantify the target. We have developed a multiplex assay based on the two-channel system for simultaneous detection and quantification of three targets in AUTO6NG CAR Tcells. The assay was highly specific, sensitive, accurate, and reproducible across time and samples. No differences were observed in measuring VCN between standard duplex and multiplex assays. Our results demonstrate that ddPCR is an accurate and cost-effective method for simultaneous detection of multiple targets in genomic DNA derived from engineered CAR Tcells.

SIMULTANEOUS PATHOGEN DETECTION OF SHRIMP VIRUSES ON CULTURED TIGER SHRIMPS (Penaeus monodon) IN INDONESIA

The multiple-pathogen infection causes severe economic impact to shrimp industry in Indonesia and worldwide due to mass mortality and multiple abnormalities of the survived infected shrimps. However, multiple-pathogen detection tools in shrimp diseases have not yet widely used. The purpose in this study was to develop and applied simultaneous detection system using multiplex polymerase chain reaction (PCR) assay from natural infections caused by white spot syndrome virus (WSSV), infectious hypodermal and haematopoietic necrosis virus (IHHNV) and monodon baculovirus (MBV) in Black tiger shrimp culture. To analyze multiple-pathogen infections in the shrimp, the study designed and used three pairs of specific primers targeting DNA virus from the shrimp diseases. All amplifications used a specific master mix for multiplex PCR assay and standardized extracted nucleic acid from the samples. This mPCR assay successfully amplified the DNA of three viruses in a single tube-run by multiplex PCR for each virus. Based on the results, the study confirms that multiple-pathogen infection contributes the highest mass mortality rather than from single infection by either WSSV, IHHNV or MBV. This study also confirms that the mPCR assay is a faster, cheaper, and efficient method to detect and subsequently prevent the spreading of multi-pathogen shrimp diseases.

Taking advance of isotropic-to-anisotropic morin-modified silver nanoparticles for simultaneous colorimetric sensing of trivalent chromium and iron ions

Taking into account colorimetric sensing purposes of silver nanoparticles (AgNPs), a convenient system for simultaneous detection of multiple metal ions has been more challenging and less exploited. In the present work, at the same condition (involving AgNPs and morin concentrations, pH and incubation time), morin-modified AgNPs were firstly applied to the simultaneous detection of Cr3+ and Fe3+ ions by naked eyes and ultraviolet-visible spectrophotometry. Furthermore, method validation was also carried out involving specification, selectivity, limits of detection and quantification. The colorimetric sensing mechanisms for such metal ions were admitted via an anisotropic growth of AgNPs, resulting in either larger aggregates due to a coordination complex (Cr3+ ion) or a smaller size due to a redox cycle (Fe3+ ion).

CRISPR-Cas12a powered multifunctional DNA nanodumbbell lock biosensor for multiple molecular detection

Detection of multiple biomarkers for accurate diagnosis of disease is highly desirable but remains challenging. Here, we developed a versatile diagnostic biosensor (SMARTLOCK) through the unique combination of DNA tetrahedron, nonlinear HCR and CRISPR-Cas12a system for simultaneous detection of multiple nucleic acids, small molecules, proteins and different types of combinations. Two DNA tetrahedrons are assembled into a functional dumbbell lock, which was unlocked by the target molecules, thereby triggering a two-stage signal amplification of nonlinear HCR and CRISPR-Cas12a to generate powerful fluorescence signals. The capability of multi-target simultaneous detection of SMARTLOCK is demonstrated in the detection of miR-141, miR-375, miR-21, ATP, cortisol, insulin and their combinations. SMARTLOCK could selectively (specificity = 93.3%) and sensitively (sensitivity = 90%) distinguish early-stage prostate cancer (PCa) patients (n = 10) from healthy donors (n = 30). Measurement of two PCa-related RNAs (miR-141 and miR-375) shows the potential of SMARTLOCK to be a highly accurate and low-cost tool for the screening, diagnosis and prognosis of various diseases.

Microfluidic-LAMP chip for the point-of-care detection of gene-deleted and wild-type African swine fever viruses and other four swine pathogens.

Different pathogens causing mixed infection are now threatening the pig industry in the context of the African Swine Fever (ASF) circulating especially in China, and it is crucial to achieving the early diagnosis of these pathogens for disease control and prevention. Here we report the development of a rapid, portable, sensitive, high-throughput, and accurate microfluidic-LAMP chip detection system for simultaneous detection and differentiation of gene-deleted type and wild-type African swine fever virus (ASFV), pseudorabie virus (PRV), porcine parvovirus (PPV), porcine circovirus type 2 (PCV2), and porcine reproductive and respiratory syndrome (PRRSV). The newly developed system was shown to be sensitive with detection limits of 101 copies/μl for ASFV-MGF505-2R/P72, PPV, and PCV2, 102 copies/μl for ASFV-CD2v, PRV, and PRRSV. The system was highly specific (100%) and stable (C.V.s < 5%) in its ability to detect different pathogens. A total 213 clinical samples and 15 ASFV nucleic acid samples were collected to assess the performance of the detection system, showing highly effective diagnosis. Altogether, the developed microfluidic-LAMP chip system provides a rapid, sensitive, high-throughput and portable diagnostic tool for the accurate detection of multiple swine pathogens.

A novel approach for simultaneous detection of the most common food-borne pathogens by multiplex qPCR.

Food contaminated with bacterial pathogens is a great threat to human health and food spoilage, having an impact on public health and the food industry. Research in food safety seeks to develop a practical, rapid, and sensitive detection technique for food-borne pathogens. In the past few decades, real-time quantitative polymerase chain reaction (qPCR) has been developed, and multiplex qPCR is a preferred feature. Multiplex qPCR enables the simultaneous amplification of many targets of interest in one reaction by using more than one pair of primers. In this study, we have developed and evaluated a hydrolysis (TaqMan) probe-based system for simultaneous detection of eight of the most common food-borne pathogens in a single-step procedure by multiplex qPCR. A multicolor combinational probe coding (MCPC) strategy was utilized that allows multiple fluorophores to label different probes in combinatorial manner. This strategy enabled simultaneous detection, identification, and quantification of targeted genes. The efficiency of the individual qPCR reactions for each target gene had values comparable to those established for multiplex qPCR, with detection limits of approximately < 10 copies of DNA per reaction. Pathogen load helps to predict bacteriological quality status in food products and serves to validate the efficiency of procedures to minimize or eliminate their presence, so newly developed multiplex qPCR was quantitative for each pathogen. During sample preparation, a step to concentrate the target organism from a relatively large sample size, remove all potential PCR inhibitors, and yield samples in a volume suitable for qPCR was incorporated.

A novel approach for simultaneous detection of the most common food-borne pathogens by multiplex qPCR.

Food contaminated with bacterial pathogens is a great threat to human health and food spoilage, having an impact on public health and the food industry. Research in food safety seeks to develop a practical, rapid, and sensitive detection technique for food-borne pathogens. In the past few decades, real-time quantitative polymerase chain reaction (qPCR) has been developed, and multiplex qPCR is a preferred feature. Multiplex qPCR enables the simultaneous amplification of many targets of interest in one reaction by using more than one pair of primers. In this study, we have developed and evaluated a hydrolysis (TaqMan) probe-based system for simultaneous detection of eight of the most common food-borne pathogens in a single-step procedure by multiplex qPCR. A multicolor combinational probe coding (MCPC) strategy was utilized that allows multiple fluorophores to label different probes in combinatorial manner. This strategy enabled simultaneous detection, identification, and quantification of targeted genes. The efficiency of the individual qPCR reactions for each target gene had values comparable to those established for multiplex qPCR, with detection limits of approximately < 10 copies of DNA per reaction. Pathogen load helps to predict bacteriological quality status in food products and serves to validate the efficiency of procedures to minimize or eliminate their presence, so newly developed multiplex qPCR was quantitative for each pathogen. During sample preparation, a step to concentrate the target organism from a relatively large sample size, remove all potential PCR inhibitors, and yield samples in a volume suitable for qPCR was incorporated.

Synthesis of fluorescent artificial receptors with high specificity for simultaneous detection of non-steroidal anti-inflammatory drugs

Development of multiple detection methods to monitor non-steroidal anti-inflammatory drugs (NSAIDs) in food is an effective way to protect human health. Here, we aimed to synthesize fluorescent artificial receptors by molecular imprinting technique to construct a simultaneous detection system targeting NSAIDs. Rhodamine B and fluorescein-functionalized silanes were employed as the fluorescence signal reporters for naproxen and ketoprofen, respectively. Two fluorescent molecularly imprinted polymers (FMIPs) were obtained with high specificity, giving cross-reactivity factors of 6.4–15.8 (naproxen) and 2.6–25.6 (ketoprofen). Both FMIPs also displayed rapid response time (5 min) and high sensitivity (detection limit at ∼ nM level). A simultaneous detection system was constructed based on the FMIPs and applied for sensing the spiked NSAIDs in real samples, showing recoveries of 71–119 %, comparable with the HPLC methods (70–113 %). In summary, use of different FMIPs to construct simultaneous detection systems is practicable, and provides a flexible way for sensing multiple hazards in food samples.

Application of Duplex Droplet Digital PCR Detection of miR-888 and miR-891a in Semen Identification.

To establish a system for simultaneous detection of miR-888 and miR-891a by droplet digital PCR (ddPCR), and to evaluate its application value in semen identification. The hydrolysis probes with different fluorescence modified reporter groups were designed to realize the detection of miR-888 and miR-891a by duplex ddPCR. A total of 75 samples of 5 body fluids (including peripheral blood, menstrual blood, semen, saliva and vaginal secretion) were detected. The difference analysis was conducted by Mann-Whitney U test. The semen differentiation ability of miR-888 and miR-891a was evaluated by ROC curve analysis and the optimal cut-off value was obtained. There was no significant difference between the dual-plex assay and the single assay in this system. The detection sensitivity was up to 0.1 ng total RNA, and the intra- and inter-batch coefficients of variation were less than 15%. The expression levels of miR-888 and miR-891a detected by duplex ddPCR in semen were both higher than those in other body fluids. ROC curve analysis showed that the AUC of miR-888 was 0.976, the optimal cut-off value was 2.250 copies/μL, and the discrimination accuracy was 97.33%; the AUC of miR-891a was 1.000, the optimal cut-off value was 1.100 copies/μL, and the discrimination accuracy was 100%. In this study, a method for detection of miR-888 and miR-891a by duplex ddPCR was successfully established. The system has good stability and repeatability and can be used for semen identification. Both miR-888 and miR-891a have high ability to identify semen, and the discrimination accuracy of miR-891a is higher.

Ultra-sensitive detection of multiplexed heavy metal ions by MOF-derived carbon film encapsulating BiCu alloy nanoparticles in potable electrochemical sensing system

In this work, we report the development of a new type of highly active and stable Bi-based electrode material, i.e., BiCu metal-organic frames (MOF) derived carbon film (CF) encapsulating BiCu alloy nanoparticles (BiCu-ANPs) for electrochemical sensing. The integration of Bi with Cu to form BiCu-ANPs can improve their electrocatalytic activity as well as the acid resistance. Meanwhile, the carbon film that encapsulates BiCu-ANPs not only guarantees the BiCu-ANPs with high electrical conductivity and fast electrochemical kinetics but also effectively alleviates the volume change during the adsorption and desorption of heavy metal (HM) ions. Therefore, the as-obtained CF encapsulating BiCu-ANPs (BiCu-ANPs@CF) exhibits fully exposed active sites, facile charge transfer, high stability and conductivity, which gives rise to enhanced sensitivity and stability for the electrochemical detection of HM ions. When integrated into a potable electrochemical sensing system for simultaneous detection of Pb2+, Cd2+ and Zn2+, the BiCu-ANPs@CF modified electrode exhibits low detection limit (i.e., 0.081 ppb for Pb2+, 0.95 ppb for Cd2+, 35 ppb for Zn2+), wide detection range (i.e., 0.5–700 ppb for Pb2+, 5–900 ppb for Cd2+, 150–600 ppb for Zn2+) and good anti-interference. Finally, the system has been used for on-site detection of multiplexed HM ions in human biological liquids and environmental water with a good spiked recovery rate, which demanstrates its promise application in the future for on-site monitoring of human health and pollutants in water quality.

Simultaneous detection of three chemical species (NO, O, O2) using a single broadband femtosecond laser

Simultaneous nitric oxide (NO) laser-induced fluorescence (LIF), atomic oxygen (O) two-photon LIF (TPLIF), and molecular oxygen (O2) LIF were demonstrated using a single broadband femtosecond (fs) laser in methane- and hydrogen-fueled flames. An amplified Ti:Sapphire laser with ∼80-fs pulse duration was used to pump an optical parametric amplifier (OPA) to generate 226.1-nm pulses having 2.3 nm (∼450 cm−1) bandwidth to excite all three species simultaneously. The specific excitation transitions are NO A-X (0,0) system, O 3p3P←←2p3P two-photon transition, and O2B3Σu−−X3Σg− Schumann-Runge system. Detailed emission characterization was performed using a 1D imaging spectrometer to identify suitable emission bands for interference-free detection of each species. A simultaneous detection system consisting of a visible and a UV camera provided the optimal detection of all three species. Imaging studies revealed high concentrations of NO, O, and O2 at the edge of the flame cone of Bunsen flames because NO and O are produced at the hot flame front. O2 LIF signal was present only at the flame front because of the strong temperature dependence of the O2 Schumann-Runge system. Direct imaging of O TPLIF enabled single-shot as well as high-fidelity shot-averaged line images in stable laminar flames. Equivalence ratio scans in the CH4/air Hencken flames showed good agreements for O and O2 with Cantera equilibrium predictions performed using GRI-Mech 3.0. Although the NO measurements deviated from the equilibrium calculations on the fuel-rich side, it agreed well with previously reported NO LIF measurements in a similar flame. This discrepancy is likely due to Prompt NO, which was not considered in the simulation. NO LIF measurements as a function of height-above-the-burner in the H2/air Hencken flames agreed well with calculated NO mole fractions using the UNICORN flame code. The present study lays the foundation for single-laser imaging of three critical flame species in NO formation pathways in combustion systems.

Simple Amplifier Coupled with a Lanthanide Labeling Strategy for Multiplexed and Specific Quantification of MicroRNAs.

Inductively coupled plasma-mass spectrometry (ICP-MS) with elemental labeling is a promising strategy for multiplex microRNA (miRNA) analysis. However, it is still challenging for specific analysis of multiple miRNAs with high homology, and the development of multiplex assays is always limited by the complexity of the sequence design. Herein, a simple and direct ICP-MS-based assay was developed for the simultaneous detection of three miRNAs by combining the lanthanide labeling strategy with entropy-driven catalytic (EDC) amplification. Owing to the specificity of EDC for nucleic acid recognition, it is able to differentiate miRNAs with single-base mutation in each EDC circuit. A universal biotin-labeled DNA strand was designed to hybridize with the DNA substrates for three EDC circuits, targeting miRNA-21, miRNA-155, and miRNA-10b, respectively. All the substrates were loaded on the surface of streptavidin magnetic beads. In the presence of target miRNA, the EDC reaction was initiated, and EDC substrates were dissociated, continuously releasing reporter strands that were labeled with lanthanides (Tb/Ho/Lu). After magnetic separation, the supernatant containing the released reporter strands was introduced into an ICP-MS system for simultaneous detection of 159Tb/165Ho/175Lu and quantification of miRNA-21, miRNA-155, and miRNA-10b, respectively. The limits of detection were 7.4, 7.5, and 11 pmol L-1 for miRNA-21, miRNA-155, and miRNA-10b, respectively. Overall, this study provides a powerful strategy for simultaneous quantification of multiple miRNAs, with the advantages of flexible probe design, good sensitivity, and excellent specificity.

An Amine-Reactive Phenazine Ethosulfate (arPES)-A Novel Redox Probe for Electrochemical Aptamer-Based Sensor.

Electrochemical aptamer-based biosensors (E-ABs) are attractive candidates for use in biomarker detection systems due to their sensitivity, rapid response, and design flexibility. There are only several redox probes that were employed previously for this application, and a combination of redox probes affords some advantages in target detection. Thus, it would be advantageous to study new redox probes in an E-AB system. In this study, we report the use of amine-reactive phenazine ethosulfate (arPES) for E-AB through its conjugation to the terminus of thrombin-binding aptamer. The constructed E-AB can detect thrombin by square-wave voltammetry (SWV), showing peak current at −0.15 V vs. Ag/AgCl at pH 7, which differs from redox probes used previously for E-ABs. We also compared the characteristics of PES as a redox probe for E-AB to methylene blue (MB), which is widely used. arPES showed stable signal at physiological pH. Moreover, the pH profile of arPES modified thrombin-binding aptamer revealed the potential application of arPES for a simultaneous multianalyte detection system. This could be achieved using different aptamers with several redox probes in tandem that harbor various electrochemical peak potentials. Our findings present a great opportunity to improve the current standard of biological fluid monitoring using E-AB.

A Dual-Channel Intelligent Point-of-Care Testing System for Soluble Programmed Death-1 and Programmed Death-Ligand 1 Detection Based on Folding Paper-Based Immunosensors.

Both programmed death-1 (PD-1) and programmed death-ligand 1 (PD-L1) are important proteins in cancer immunotherapy. Soluble forms (sPD-1 and sPD-L1) have potential for determining treatment and prognosis monitoring. However, there is a lack of detection methods for point-of-care testing (POCT) of these two proteins, so a low-cost rapid detection platform is urgently needed. To solve this problem, a dual-channel electrochemical platform, including a folding paper-based immunosensor and a POCT system for rapid simultaneous detection of these two proteins was designed and fabricated. The immunosensor consists of a three-electrode system and a reaction cell. The surface of the working electrode was modified with nanocomposites synthesized from amine-functionalized single-walled carbon nanotubes, new methylene blue, and gold nanoparticles. Antibodies to sPD-1 and sPD-L1 were also immobilized on the working electrode surface. A differential pulse voltammetry electrochemical method was adopted. The immunosensor was able to detect sPD-1 and sPD-L1 in the ranges of 50 pg/mL to 50 ng/mL and 5 pg/mL to 5 ng/mL, respectively. The limits of detection were 10 and 5 pg/mL. Using this detection platform, sPD-1 and sPD-L1 in plasma were detected by both enzyme-linked immunosorbent assay and the immunosensor, which has good application potential.