Disposable graphene-oxide screen-printed electrode integrated with portable device for detection of SARS-CoV-2 in clinical samples.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnosis is the need of the hour, as cases are persistently increasing, and new variants are constantly emerging. The ever-changing nature of the virus leading to multiple variants, has brought an imminent need for early, accurate and rapid detection methods. Herein, we have reported the design and fabrication of Screen-Printed Electrodes (SPEs) with graphene oxide (GO) as working electrode and modified with specific antibodies for SARS-CoV-2 Receptor Binding Domain (RBD). Flexibility of design, and portable nature has made SPEs the superior choice for electrochemical analysis. The developed immunosensor can detect RBD as low as 0.83 fM with long-term storage capacity. The fabricated SPEs immunosensor was tested using a miniaturized portable device and potentiostat on 100 patient nasopharyngeal samples and corroborated with RT-PCR data, displayed 94 % sensitivity. Additionally, the in-house developed polyclonal antibodies detected RBD antigen of the mutated Omicron variant of SARS-CoV-2 successfully. We have not observed any cross-reactivity/binding of the fabricated immunosensor with MERS (cross-reactive antigen) and Influenza A H1N1 (antigen sharing common symptoms). Hence, the developed SPEs sensor may be applied for bedside point-of-care diagnosis of SARS-CoV-2 using miniaturized portable device, in clinical samples.

A portable transistor immunosensor for fast identification of porcine epidemic diarrhea virus.

Widespread distribution of porcine epidemic diarrhea virus (PEDV) has led to catastrophic losses to the global pig farming industry. As a result, there is an urgent need for rapid, sensitive and accurate tests for PEDV to enable timely and effective interventions. In the present study, we develop and validate a floating gate carbon nanotubes field-effect transistor (FG CNT-FET)-based portable immunosensor for rapid identification of PEDV in a sensitive and accurate manner. To improve the affinity, a unique PEDV spike protein-specific monoclonal antibody is prepared by purification, and subsequently modified on FG CNT-FET sensor to recognize PEDV. The developed FET biosensor enables highly sensitive detection (LoD: 8.1 fg/mL and 100.14 TCID50/mL for recombinant spike proteins and PEDV, respectively), as well as satisfactory specificity. Notably, an integrated portable platform consisting of a pluggable FG CNT-FET chip and a portable device can discriminate PEDV positive from negative samples and even identify PEDV and porcine deltacoronavirus within 1 min with 100% accuracy. The portable sensing platform offers the capability to quickly, sensitively and accurately identify PEDV, which further points to a possibility of point of care (POC) applications of large-scale surveillance in pig breeding facilities.

Rapid, on-site quantitative determination of mycotoxins in grains using a multiple time-resolved fluorescent microsphere immunochromatographic test strip.

The label probe plays a crucial role in enhancing the sensitivity of lateral flow immunoassays. However, conventional fluorescent microspheres (FMs) have limitations due to their short fluorescence lifetime, susceptibility to background fluorescence interference, and inability to facilitate multi-component detection. In this study, carboxylate-modified Eu(III)-chelate-doped polystyrene nanobeads were employed as label probes to construct a multiple time-resolved fluorescent microsphere-based immunochromatographic test strip (TRFM-ICTS). This novel TRFM-ICTS facilitated rapid on-site quantitative detection of three mycotoxins in grains: Aflatoxin B1 (AFB1), Zearalenone (ZEN), and Deoxynivalenol (DON). The limit of detection (LOD) for AFB1, ZEN, and DON were found to be 0.03 ng/g, 0.11 ng/g, and 0.81 ng/g, respectively. Furthermore, the TRFM-ICTS demonstrated a wide detection range for AFB1 (0.05-8.1 ng/g), ZEN (0.125-25 ng/g), and DON (1.0-234 ng/g), while maintaining excellent selectivity. Notably, the test strip exhibited remarkable stability, retaining its detection capability even after storage at 4 °C for over one year. Importantly, the detection of these mycotoxins relied solely on simple manual operations, and with a portable reader, on-site detection could be accomplished within 20 min. This TRFM-ICTS presents a promising solution for sensitive on-site mycotoxin detection, suitable for practical application in various settings due to its sensitivity, accuracy, simplicity, and portability.

Nitrocellulose/acrylic resin coated screen-printed carbon electrode to construct a capacitive immunosensor for anti-BSA.

The capacitive immunosensor, known for its label-free simplicity, has great potential for point-of-care diagnostics. However, the interaction between insulation and recognition layers on the sensing electrode greatly affects its performance. This study introduces a pioneering dual-layer strategy, implementing a novel combination of acrylic resin (AR) and nitrocellulose (NC) coatings on screen-printed carbon electrodes (SPCEs). This innovative approach not only enhances the dielectric properties of the capacitive sensor but also streamlines the immobilization of recognizing elements. Particularly noteworthy is the superior reliability and insulation offered by the AR coating, surpassing the limitations of traditional self-assembled monolayer (SAM) modifications. This dual-layer methodology establishes a robust foundation for constructing capacitive sensors optimized specifically for liquid medium-based biosensing applications. The NC coating in this study represents a breakthrough in effectively immobilizing BSA, unraveling the capacitive response intricately linked to the quantity of adsorbed recognizing elements. The results underscore the prowess of the proposed immunosensor, showcasing a meticulously defined linear calibration curve for anti-BSA (ranging from 0 to 25 µg/ml). Additionally, specific interactions with anti-HAS and anti-TNF-α further validate the versatility and efficacy of the developed immunosensor. This work presents a streamlined and highly efficient protocol for developing label-free immunosensors for antibody determination and introduces a paradigm shift by utilizing readily available electrodes and sensing systems. The findings are poised to catalyze a significant acceleration in the advancement of biosensor technology, opening new avenues for innovative applications in point-of-care diagnostics.

Microfluidic Cartridge for Bead-Based Affinity Assays.

Within the vast field of medical biotechnology, the biopharmaceutical industry is particularly fast-growing and highly competitive, so reducing time and costs associated to process optimization becomes instrumental to ensure speed to market and, consequently, profitability. The manufacturing of biopharmaceutical products, namely, monoclonal antibodies (mAbs), relies mostly on mammalian cell culture processes, which are highly dynamic and, consequently, difficult to optimize. In this context, there is currently an unmet need of analytical methods that can be integrated at-line in a bioreactor, for systematic monitoring and quantification of key metabolites and proteins. Microfluidic-based assays have been extensively and successfully applied in the field of molecular diagnostics; however, this technology remains largely unexplored for Process Analytical Technology (PAT), despite holding great potential for the at-line measurement of different analytes in bioreactor processes, combining low reagent/molecule consumption with assay sensitivity and rapid turnaround times.Here, the fabrication and handling of a microfluidic cartridge for protein quantification using bead-based affinity assays is described. The device allows geometrical multiplexed immunodetection of specific protein analytes directly from bioreactor samples within 2.5 h and minimal hands-on time. As a proof-of-concept, quantification of Chinese hamster ovary (CHO) host cell proteins (HCP) as key impurities, IgG as product of interest, and lactate dehydrogenase (LDH) as cell viability marker was demonstrated with limits of detection (LoD) in the low ng/mL range. Negligible matrix interference and no cross-reactivity between the different immunoassays on chip were found. The results highlight the potential of the miniaturized analytical method for PAT at reduced cost and complexity in comparison with sophisticated instruments that are currently the state-of-the-art in this context.

Enhancing the Efficiency of Conventional Surface Immunoassays Within Standard Labware Using Microscale Flows.

In this chapter, we present the design and fabrication of a device and implementation of a protocol to realize increased efficiency of immunoassays within microtiter plates. The device, WellProbe, is a 3D-structured probe that can be used to deliver precise flows at the bottom of standard well plates to establish concentric areas of shear stress intensities using hydrodynamically confined flows. The protocols involve both operation and data analysis.

Rapid and on-site wireless immunoassay of respiratory virus aerosols via hydrogel-modulated resonators.

Rapid and accurate detection of respiratory virus aerosols is highlighted for virus surveillance and infection control. Here, we report a wireless immunoassay technology for fast (within 10 min), on-site (wireless and battery-free), and sensitive (limit of detection down to fg/L) detection of virus antigens in aerosols. The wireless immunoassay leverages the immuno-responsive hydrogel-modulated radio frequency resonant sensor to capture and amplify the recognition of virus antigen, and flexible readout network to transduce the immuno bindings into electrical signals. The wireless immunoassay achieves simultaneous detection of respiratory viruses such as severe acute respiratory syndrome coronavirus 2, influenza A H1N1 virus, and respiratory syncytial virus for community infection surveillance. Direct detection of unpretreated clinical samples further demonstrates high accuracy for diagnosis of respiratory virus infection. This work provides a sensitive and accurate immunoassay technology for on-site virus detection and disease diagnosis compatible with wearable integration.

Automatic ultrasensitive lateral flow immunoassay based on a color-enhanced signal amplification strategy.

Lateral flow immunoassays (LFIAs) are an essential and widely used point-of-care test for medical diagnoses. However, commercial LFIAs still have low sensitivity and specificity. Therefore, we developed an automatic ultrasensitive dual-color enhanced LFIA (DCE-LFIA) by applying an enzyme-induced tyramide signal amplification method to a double-antibody sandwich LFIA for antigen detection. The DCE-LFIA first specifically captured horseradish peroxidase (HRP)-labeled colored microspheres at the Test line, and then deposited a large amount of tyramide-modified signals under the catalytic action of HRP to achieve the color superposition. A limit of detection (LOD) of 3.9 pg/mL and a naked-eye cut-off limit of 7.8 pg/mL were achieved for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleoprotein. Additionally, in the inactivated virus detections, LOD equivalent to chemiluminescence (0.018 TCID50/mL) was obtained, and it had excellent specificity under the interference of other respiratory viruses. High sensitivity has also been achieved for detection of influenza A, influenza B, cardiac troponin I, and human chorionic gonadotrophin using this DCE-LFIA, suggesting the assay is universally applicable. To ensure the convenience and stability in practical applications, we created an automatic device. It provides a new practical option for point-of-care test immunoassays, especially ultra trace detection and at-home testing.

Polymeric optical fiber biosensor with PAMAM dendrimer-based surface modification and PlGF detection for pre-eclampsia diagnosis.

Pre-eclampsia (PE) is a life-threatening complication that occurs during pregnancy, affecting a large number of pregnant women and newborns worldwide. Rapid, on-site and affordable screening of PE at an early stage is necessary to ensure timely treatment and minimize both maternal and neonatal morbidity and mortality rates. Placental growth factor (PlGF) is an angiogenic blood biomarker used for PE diagnosis. Herein, we report the plasmonic fiber optic absorbance biosensor (P-FAB) strategy for detecting PlGF at femtomolar concentration using polymethyl methacrylate (PMMA) based U-bent polymeric optical fiber (POF) sensor probes. A novel poly(amidoamine) (PAMAM) dendrimer based PMMA surface modification is established to obtain a greater immobilization of the bioreceptors compared to a linear molecule like hexamethylenediamine (HMDA). Plasmonic sandwich immunoassay was realized by immobilizing the mouse anti-PlGF (3H1) on the U-bent POF sensor probe surface and gold nanoparticles (AuNP) labels conjugated with mouse anti-PlGF (6H9). The POF sensor probes could measure PlGF within 30 min using the P-FAB strategy. The limit-of-detection (LoD) was found to be 0.19 pg/mL and 0.57 pg/mL in phosphate-buffered saline and 10× diluted serum, respectively. The clinical sample testing, with eleven positive and eleven negative preeclamptic pregnancy samples, successfully confirmed the accuracy, reliability, specificity, and sensitivity of the P-FAB based POF sensor platform, thereby paving the way for cost-effective technology for PlGF detection and its potential for pre-eclampsia diagnosis.

Graphene Nanocomposite Ink Coated Laser Transformed Flexible Electrodes for Selective Dopamine Detection and Immunosensing.

Novel and flexible disposable laser-induced graphene (LIG) sensors modified with graphene conductive inks have been developed for dopamine and interleukin-6 (IL-6) detection. The LIG sensors exhibit high reproducibility (relative standard deviation, RSD = 0.76%, N = 5) and stability (RSD = 4.39%, N = 15) after multiple bendings, making the sensors ideal for wearable and stretchable bioelectronics applications. We have developed electrode coatings based on graphene conductive inks, poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (G-PEDOT:PSS) and polyaniline (G-PANI), for working electrode modification to improve the sensitivity and limit of detection (LOD). The selectivity of LIG sensors modified with the G-PANI ink is 41.47 times higher than that of the screen-printed electrode with the G-PANI ink modification. We have compared our fabricated bare laser-engraved Kapton sensor (LIG) with the LIG sensors modified with G-PEDOT (LIG/G-PEDOT) and G-PANI (LIG/G-PANI) conductive inks. We have further compared the performance of the fabricated electrodes with commercially available screen-printed electrodes (SPEs) and screen-printed electrodes modified with G-PEDOT:PSS (SPE/G-PEDOT:PSS) and G-PANI (SPE/G-PANI). SPE/G-PANI has a lower LOD of 0.632 µM compared to SPE/G-PEDOT:PSS (0.867 µM) and SPE/G-PANI (1.974 µM). The lowest LOD of the LIG/G-PANI sensor (0.4084 µM, S/N = 3) suggests that it can be a great alternative to measure dopamine levels in a physiological medium. Additionally, the LIG/G-PANI electrode has excellent LOD (2.6234 pg/mL) to detect IL-6. Also, the sensor is successfully able to detect ascorbic acid (AA), dopamine (DA), and uric acid (UA) in their ternary mixture. The differential pulse voltammetry (DPV) result shows peak potential separation of 229, 294, and 523 mV for AA-DA, DA-UA, and UA-AA, respectively.

"Cave Effect" Induces Self-Assembled Bimetallic Hollow Structure for Three-in-One Lateral Flow Immunoassay.

Bimetallic hollow structures have attracted much attention due to their unique properties, but they still face the problems of nonuniform alloys and excessive etching leading to structural collapse. Here, uniform bimetallic hollow nanospheres are constructed by pore engineering and then highly loaded with hemin (Hemin@MOF). Interestingly, in the presence of polydopamine (PDA), the competitive coordination between anionic polymer (γ-PGA) and dimethylimidazole does not lead to the collapse of the external framework but self-assembly into a hollow structure. By constructing the Hemin@MOF immune platform and using E. coli O157:H7 as the detection object, we find that the visual detection limits can reach 10, 3, and 3 CFU/mL in colorimetric, photothermal, and catalytic modes, which is 4 orders of magnitude lower than the traditional gold standard. This study provides a new idea for the morphological modification of the metal-organic skeleton and multifunctional immunochromatography detection.

Sample-to-answer lateral flow assay with integrated plasma separation and NT-proBNP detection.

Through enabling whole blood detection in point-of-care testing (POCT), sedimentation-based plasma separation promises to enhance the functionality and extend the application range of lateral flow assays (LFAs). To streamline the entire process from the introduction of the blood sample to the generation of quantitative immune-fluorescence results, we combined a simple plasma separation technique, an immunoreaction, and a micropump-driven external suction control system in a polymer channel-based LFA. Our primary objective was to eliminate the reliance on sample-absorbing separation membranes, the use of active separation forces commonly found in POCT, and ultimately allowing finger prick testing. Combining the principle of agglutination of red blood cells with an on-device sedimentation-based separation, our device allows for the efficient and fast separation of plasma from a 25-µL blood volume within a mere 10 min and overcomes limitations such as clogging, analyte adsorption, and blood pre-dilution. To simplify this process, we stored the agglutination agent in a dried state on the test and incorporated a filter trench to initiate sedimentation-based separation. The separated plasma was then moved to the integrated mixing area, initiating the immunoreaction by rehydration of probe-specific fluorophore-conjugated antibodies. The biotinylated immune complex was subsequently trapped in the streptavidin-rich detection zone and quantitatively analyzed using a fluorescence microscope. Normalized to the centrifugation-based separation, our device demonstrated high separation efficiency of 96% and a yield of 7.23 µL (= 72%). Furthermore, we elaborate on its user-friendly nature and demonstrate its proof-of-concept through an all-dried ready-to-go NT-proBNP lateral flow immunoassay with clinical blood samples.

Centrifugo-Pneumatic Reciprocating Flowing Coupled with a Spatial Confinement Strategy for an Ultrafast Multiplexed Immunoassay.

Immunoassays serve as powerful diagnostic tools for early disease screening, process monitoring, and precision treatment. However, the current methods are limited by high costs, prolonged processing times (>2 h), and operational complexities that hinder their widespread application in point-of-care testing. Here, we propose a novel centrifugo-pneumatic reciprocating flowing coupled with spatial confinement strategy, termed PRCM, for ultrafast multiplexed immunoassay of pathogens on a centrifugal microfluidic platform. Each chip consists of four replicated units; each unit allows simultaneous detection of three targets, thereby facilitating high-throughput parallel analysis of multiple targets. The PRCM platform enables sequential execution of critical steps such as solution mixing, reaction, and drainage by coordinating inherent parameters, including motor rotation speed, rotation direction, and acceleration/deceleration. By integrating centrifugal-mediated pneumatic reciprocating flow with spatial confinement strategies, we significantly reduce the duration of immune binding from 30 to 5 min, enabling completion of the entire testing process within 20 min. As proof of concept, we conducted a simultaneous comparative test on- and off-the-microfluidics using 12 negative and positive clinical samples. The outcomes yielded 100% accuracy in detecting the presence or absence of the SARS-CoV-2 virus, thus highlighting the potential of our PRCM system for multiplexed point-of-care immunoassays.

Quantitative measurement of acute myocardial infarction cardiac biomarkers by "All-in-One" immune microfluidic chip for early diagnosis of myocardial infarction.

Acute myocardial infarction (AMI) is a life-threatening condition with a narrow treatment window, necessitating rapid and accurate diagnostic methods. We present an "all-in-one" convenient and rapid immunoassay system that combines microfluidic technology with a colloidal gold immunoassay. A degassing-driven chip replaces a bulky external pump, resulting in a user-friendly and easy-to-operate immunoassay system. The chip comprises four units: an inlet reservoir, an immunoreaction channel, a waste pool, and an immunocomplex collection chamber, allowing single-channel flow for rapid and accurate AMI biomarker detection. In this study, we focused on cardiac troponin I (cTnI). With a minimal sample of just 4 µL and a total detection time of under 3 min, the chip enabled a quantitative visual analysis of cTnI concentration within a range of 0.5 âˆ¼ 60.0 ng mL-1. This all-in-one integrated microfluidic chip with colloidal gold immunoassay offers a promising solution for rapid AMI diagnosis. The system's portability, small sample requirement, and quantitative visual detection capabilities make it a valuable tool for AMI diagnostics.

A metal-organic framework signaling probe-mediated immunosensor for the economical and rapid determination of enrofloxacin in milk.

Ensuring the safety of animal-derived foods requires the reliable and swift identification of enrofloxacin residues to monitor the presence of antibiotics. In this regard, we synthesized, tuned, and investigated the optical properties of a bimetallic metal-organic framework (Ce/Zr-UiO 66). The investigation was facilitated by employing a polydopamine-coated pipette tip with high adsorption efficiency, serving as an immunoreactive carrier. Subsequently, an immunofunctionalized variant of Ce/Zr-UiO 66, referred to as Ce/Zr-UiO 66@ Bovine serum albumin-enrofloxacin, was developed as an optical probe for the rapid and sensitive identification of enrofloxacin across a variety of samples. The method can accurately detect enrofloxacin at concentrations as low as 0.12 ng/mL, with a determination time of under 15 min; furthermore, it demonstrates exceptional efficacy when applied to food, environmental, and clinical samples. The implementation of this methodology offers a valuable means for cost-effective, rapid, and on-site enrofloxacin determination.

Integration of a biocompatible metal-phenolic network and fluorescence microspheres as labels for sensitive and stable detection of carbendazim with a lateral flow immunoassay.

High fluorescence intensity microspheres such as aggregation-induced emission fluorescence microspheres (AIEFM) have improved the sensitivity of lateral flow immunoassay (LFIA). The preparation of immune probes in LFIA usually adopts the chemical coupling strategy with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide for antibody coupling, which has the problems of low coupling efficiency, tedious coupling process, and poor repeatability. A biocompatible metal-phenolic network (MPN), which contains large amounts of phenols and galloyl groups, could easily, quickly, and stably couple with antibodies. Herein, we proposed a strategy based on MPN modification on ultrabright AIEFM surface as a novel label for the rapid detection of carbendazim. The limit of detection of AIEFM@MPN-LFIA was 0.019 ng/mL, which was 4.9 times lower than that of AIEFM-LFIA. In spiked samples, the average recoveries of AIEFM@MPN-LFIA ranged from 80% to 118% (coefficient of variation <13.45%). Therefore, AIEFM@MPN was a promising signal label that could improve the detection performance of LFIA.

Novel litchi-like Au-Ag nanospheres driven dual-readout lateral flow immunoassay for sensitive detection of pyrimethanil.

Pyrimethanil (PYR) is a fungicide that is harmful to consumers when present in foods at concentrations greater than maximum permitted residue levels. High-performance immunoprobes and dual-readout strategy may be useful for constructing sensitive lateral flow immunoassay (LFIA). Herein, the prepared litchi-like Au-Ag bimetallic nanospheres (LBNPs) exhibited high mass extinction coefficients and fluorescence quenching constants. Benefiting from LBNPs and dual-readout mode, the limits of detection of LBNPs-CM-LFIA and LBNPs-FQ-LFIA for PYR were 0.957 and 0.713 ng mL-1, which were 2.54- and 3.41-fold lower than that of gold nanoparticles-based LFIA, respectively. The limits of quantitation of LBNPs-CM-LFIA and LBNPs-FQ-LFIA were 3.740 and 1.672 ng mL-1, respectively. LBNPs-LFIA was applied to detect PYR in cucumber and grape samples with satisfactory recovery (90%-111%). LBNPs-LFIA showed good agreement with LC-MS/MS for the detection of PYR in the samples. Accordingly, this sensitive and accurate dual-readout LFIA based on LBNPs can be effectively applied for food safety.

Vanadium-doped metal-organic framework@Znln2S4 core-shell heterojunction-attenuated photoelectrochemical immunoassay.

Considering that cancer has become the second leading cause of death in humans, it is essential to develop an analytical approach that can sensitively detect tumor markers for early detection. We report an attenuated photoelectrochemical (PEC) immunoassay based on the organic-inorganic heterojunction 10MIL-88B(FeV)/ZnIn2S4 (10M88B(FeV)/ZIS) as a photoactive material for monitoring carcinoembryonic antigen (CEA). The 10M88B(FeV)/ZIS heterojunctions have excellent light-harvesting properties and high electrical conductivity, which are attributed to the advantages of both organic and inorganic semiconductors, namely, remarkable photogenerated carrier separation efficiency and long photogenerated carrier lifetime. Horseradish peroxidase (HRP) in the presence of H2O2 can catalyze 3,3'-diaminofenamide (DAB) producing brown precipitates (oxDAB), which is then loaded onto the 10M88B(FeV)/ZIS heterojunction to reduce the photocurrent and enable the quantitative detection of CEA. Under optimal conditions, the photocurrent values of the PEC biosensor are linearly related to the logarithm of the CEA concentrations, ranging from 0.01 ng mL-1 to 100 ng mL-1 with a detection limit (LOD) of 4.0 pg mL-1. Notably, the accuracy of the PEC biosensor is in agreement with that of the human CEA enzyme-linked immunosorbent assay (ELISA) kit.

Construction of AlGaN/GaN high-electron-mobility transistor-based biosensor for ultrasensitive detection of SARS-CoV-2 spike proteins and virions.

The COVID-19 pandemic has highlighted the need for rapid and sensitive detection of SARS-CoV-2. Here, we report an ultrasensitive SARS-CoV-2 immunosensor by integration of an AlGaN/GaN high-electron-mobility transistor (HEMT) and anti-SARS-CoV-2 spike protein antibody. The AlGaN/GaN HEMT immunosensor has demonstrated the capability to detect SARS-CoV-2 spike proteins at an impressively low concentration of 10-22 M. The sensor was also applied to pseudoviruses and SARS-CoV-2 ΔN virions that display the Spike proteins with a single virion particle sensitivity. These features validate the potential of AlGaN/GaN HEMT biosensors for point of care tests targeting SARS-CoV-2. This research not only provides the first HEMT biosensing platform for ultrasensitive and label-free detection of SARS-CoV-2.

Three-Dimensional Microfluidic Capillary Device for Rapid and Multiplexed Immunoassays in Whole Blood.

In this study, we demonstrate whole blood immunoassays using a microfluidic device optimized for conducting rapid and multiplexed fluorescence-linked immunoassays. The device is capable of handling whole blood samples without any preparatory treatment. The three-dimensional channels in poly(methyl methacrylate) are designed to passively load bodily fluids and, due to their linearly tapered profile, facilitate size-dependent immobilization of biofunctionalized particles. The channel geometry is optimized to allow for the unimpeded flow of cellular constituents such as red blood cells (RBCs). Additionally, to make the device easier to operate, the biofunctionalized particles are pretrapped in a first step, and the channel is dried under vacuum, after which it can be loaded with the biological sample. This novel approach and design eliminated the need for traditionally laborious steps such as filtering, incubation, and washing steps, thereby substantially simplifying the immunoassay procedures. Moreover, by leveraging the shallow device dimensions, we show that sample loading to read-out is possible within 5 min. Our results also show that the presence of RBCs does not compromise the sensitivity of the assays when compared to those performed in a pure buffer solution. This highlights the practical adaptability of the device for simple and rapid whole-blood assays. Lastly, we demonstrate the device's multiplexing capability by pretrapping particles of different sizes, each functionalized with a different antigen, thus enabling the performance of multiplexed on-chip whole-blood immunoassays, showcasing the device's versatility and effectiveness toward low-cost, simple, and multiplexed sensing of biomarkers and pathogens directly in whole blood.