Multi-step Assay Research Articles

Drop-by-Drop Addition of Reagents to a Double Emulsion.

Developments in droplet microfluidics have facilitated an era of high-throughput, sensitive single-cell, or single-molecule measurements capable of tackling the heterogeneity present in biological systems. Relying on single emulsion (SE) compartments, droplet assays achieve absolute quantification of nucleic acids, massively parallel single-cell profiling, and more. Double emulsions (DEs) have seen recent interest for their potential to build upon SE techniques. DEs are compatible with flow cytometry enabling high-throughput multi-parameter drop screening and eliminate content mixing due to coalescence during lengthy workflows. Despite these strengths, DEs lack important technical functions that exist in SEs such as methods for adding reagents to droplets on demand. Consequently, DEs cannot be used for multistep workflows which has limited their adoption in assay development. Here, strategies to enable reagent addition and other active manipulations on DEs are reported by converting DE inputs to SEs on chip. After conversion, drops are manipulated using existing SE techniques, including reagent addition, before reforming a DE at the outlet. Device designs and operation conditions achieving drop-by-drop reagent addition to DEs are identified and used as part of a multi-step aptamer screening assay performed entirely in DE drops. This work enables the further development of multistep DE droplet assays.

Abstract 5566: Streamlining tissue sample preparation: A comparative study on the efficiency and reproducibility of the Omni-Stainer™ dewaxing and HIER protocol

Abstract The evolution of life sciences research has entailed a shift toward understanding cellular functions within tissue microenvironments, with an emphasis on cell- and tissue-specific relationships. A key challenge in these endeavors is the need for accurate data acquisition, which can be impeded by variability in the multistep manual assay preparations. In the quest for developing reliable and efficient tissue sample preparation workflows, two protocols using multiple dewaxing agents have been compared: the Omni-Stainer™ Dewaxing and HIER antigen retrieval protocol from Parhelia Biosciences, Inc. and the analogous one on Leica BOND Rx. The objective of this research was to assess the efficiency and reproducibility of these methods in the context of a four-color morphology immunostaining used for GeoMx DSP RNA and protein detection assays (Nanostring). We examined the efficiency of automated dewaxing using various non-toxic xylene alternatives, including C12-15 alkyl Benzoate, diethylene glycol, methyl octanoate or methyl decanoate, and compared them to the proprietary Leica Bond Dewaxing solution. Then, we compared the quality of post-HIER immunostaining using two different four-antibody panels on human jejunum samples. After several rounds of optimization, we found that the V4 of the open-source Omni-Stainer™ dewax-HIER protocol resulted in identical quality of morphology immunostaining compared to proprietary Leica Bond protocol. This study validates the Omni-Stainer™ Dewaxing and HIER protocol as a viable and efficient approach for automation of sample prep for GeoMx DSP studies. Citation Format: Nikolay Samusik, Yury Goltsev, Noemi Kedei. Streamlining tissue sample preparation: A comparative study on the efficiency and reproducibility of the Omni-Stainer™ dewaxing and HIER protocol [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5566.

Enhancement of telomerase extension via quadruple nucleic acid recycling to develop a novel colorimetric biosensing method for kanamycin assay

BackgroundColorimetric biosensors have important value for antibiotic residue testing. However, many previous methods were constructed based on the optical density change of certain unstable single-colored products with poor discrimination for visual measurements. Moreover, their low extinction coefficients usually result in low sensitivity of biosensors. In addition, many conventional signal amplification strategies often involve sophisticated nanomaterial preparation, inconvenient multi-step assay manipulation and limited signal amplification ability. Therefore, the development of new colorimetric biosensing strategies with excellent visual discrimination, high sensitivity and convenient manipulation is highly desirable. ResultsWe designed a target recycling accelerated cascade DNA walking amplification mechanism to trigger a telomerase extension-related enzymatic reaction, and developed a novel colorimetric biosensing strategy for kanamycin (Kana) assay. The target recycling was induced by an exonuclease III-assisted aptamer recognition reaction, which could also trigger the successive DNA walking at the streptavidin (SA)- and magnetic bead (MB)-based tracks. This not only caused the quantitative exposure of the telomeric substrate primers on MB surfaces but also released another strand to accelerate the SA-based DNA walking. By using the telomerase extension product to link numerous alkaline phosphatases and induce the plasmonic property change of gold nanobipyramids (Au NBPs), a colorimetric signal output strategy was constructed. This method could be applied for the high-resolution visual screening of Kana, and it also showed a very low detection limit of 17.6 fg mL−1 for assaying Kana over a wide, five-order-magnitude linear range. SignificanceThe quadruple nucleic acid recycling-enhanced telomerase extension resulted in the ultrahigh sensitivity of the method and also excluded the sophisticated manipulations involved in conventional biosensing strategies. The multiple enzyme catalysis-induced plasmonic property change of Au NBPs realized the stable and multicolor visual signal transduction. Together with its low cost, simple operation, high selectivity, excellent repeatability, and reliable performances, this method exhibits great potential for use in practical applications.

Rapid and label-free screening of SARS-CoV-2 virus in the infected human nasopharyngeal swab samples using UiO-66-modified SPR chip

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus has become a dangerous human pathogen and caused severe illness that can lead to death. Yet, the standard practical diagnostic tools for detecting the SARS-CoV-2 virus require multi-step assay, including extraction and labeling steps. Thus, a simple and accurate detection tool is incredibly desirable to support on-site screening, preventing its spread. In this work, we developed a portable surface plasmon resonance (SPR) sensor chip modified with metal–organic frameworks (MOF) UiO-66 that serves as an immobilization matrix of single-chain variable fragment (scFv) receptors. Large surface area and good adsorption of UiO-66/scFv-modified surface lead to an improvement of binding capacity towards the protein target of the SARS-CoV-2 receptor binding domain (RBD). We further successfully demonstrated the use of this portable SPR to detect the SARS-CoV-2 virus from patients' nasopharyngeal swab samples. Interestingly, the SPR sensors can directly distinguish the positive and negative patients within 15 minutes on 23 samples, simultaneously, without the need for labeling, and the results are in line with the gold standard PCR tests. In addition, the detection limit of the sensors is 10.085 × 105 virus/mL, which follows the standard of practical screening tools. Therefore, these UiO-66 modified SPR chip sensors offer a rapid, label-free, and low-cost detection of the SARS-CoV-2 virus that can be potentially developed for other infectious diseases.

A gravity-driven droplet fluidic point-of-care test

We report a simple droplet fluidic point-of-care test (POCT) that uses gravity to manipulate the sequence, timing, and motion of droplets on a surface. To fabricate this POCT, we first developed a surface coating toolbox of nine different coatings with three levels of wettability and three levels of slipperiness that can be independently tailored. We then fabricated a device that has interconnected fluidic elements-pumps, flow resistors and flow guides-on a highly slippery solid surface to precisely control the timing and sequence of motion of multiple droplets and their interactions on the surface. We then used this device to carry out a multi-step enzymatic assay of a clinically relevant analyte-lactate dehydrogenase (LDH)-to demonstrate the application of this technology for point-of-care diagnosis.

Electrochemical biosensors based on saliva electrolytes for rapid detection and diagnosis.

In recent years, electrochemical biosensors (ECBSs) have shown significant potential for real-time disease diagnosis and in situ physical condition monitoring. As a multi-constituent oral fluid comprising various disease signaling biomarkers, saliva has drawn much attention in the field of point-of-care (POC) testing. In particular, during the outbreak of the COVID-19 pandemic, ECBSs which hold the simplicity of a single-step assay compared with the multi-step assay of traditional testing methods are expected to relieve the human and economic burden caused by the massive and long-term sample testing process. Noteworthily, ECBSs for the detection of SARS-CoV-2 in saliva have already been developed and may replace current testing methods. Furthermore, the detection scope has expanded from routine indices such as sugar and uric acid to abnormal biomarkers for early-stage disease detection and drug level monitoring, which further facilitated the evolution of ECBSs in the last 5 years. This review is divided into several main sections. First, we discussed the latest advancements and representative research on ECBSs for saliva testing. Then, we focused on a novel kind of ECBS, organic electrochemical transistors (OECTs), which hold great advantages of high sensitivity and signal-to-noise ratio and on-site detection. Finally, application of ECBSs with integrated portable platforms in oral cavities, which lead to powerful auxiliary testing means for telemedicine, has also been discussed.

Dissolvable Film-Controlled Buoyancy Pumping and Aliquoting on a Lab-On-A-Disc

Lab-on-a-Disc (LoaD) has great potential for applications in decentralised bioanalytical testing where speed and robustness are critical. Here, a disc-shaped microfluidic chip is rotated to pump liquid radially outwards; thus, all microfluidic structures must be fitted into the available radial length. To overcome this limitation, several centripetal pumping technologies have been developed. In this work, we combine buoyancy pumping, enabled by displacing aqueous samples and reagents centripetally inwards by a dense liquid (fluorocarbon FC-40), with dissolvable film (DF) to automate a multi-step assay. The DF dissolves in the presence of water but is not in contact with the FC-40. Therefore, the FC-40 can be stored behind the DF membranes and is autonomously released by contact with the arriving aqueous sample. Using this technology, tasks such as blood centrifugation can be located on the disc periphery where ‘disc real estate’ is less valuable and centrifugal forces are higher. To demonstrate this, we use the combination of the buoyancy-driven centripetal pumping with DF barriers to implement a fully automated multi-parameter diagnostic assay on the LoaD platform. The implemented steps include plasma extraction from a structure, automatically triggered metering/aliquoting, and the management of five onboard stored liquid reagents. Critically, we also demonstrate highly accurate aliquoting of reagents using centripetal pumping. We also provide a mathematical model to describe the pumping mechanism and apply lumped-element modelling and Monte Carlo simulation to estimate errors in the aliquoting volumes caused by manufacturing deviations.

Multifunctional rotational active valve for flow control in paper-based microfluidic devices

In paper-based microfluidics, the development of a robust and multi-role valve mechanism is important for flow control to perform multistep biochemical assays. The previous valve designs were complex, limited in their operational capabilities to simply ON/OFF functionality, required complex circuitry and auxiliary devices for valve actuation, or were unfit for friendly user operation. Herein, we present a multifunctional, simple, robust and cost effective rotational active valve (RAV) for paper-based microfluidics devices. In addition to ON/OFF, it achieves variable flow velocity by varying the contact area of RAV channel, by varying the RAV channel length, or by changing the RAV channel width. The functionality was demonstrated in paper-based microfluidic devices for sequential delivery with intermixing, multi-reagent valve regulation with washing step, and fluid distribution to multiple delivery zones. Finally, we conducted a multistep biochemical assay for the detection of total protein, uric acid and albumin protein in human serum at various concentration (dilution ranges from 40% to 100%) using RAV. We believe that our RAV can solve complex biochemical assays in paper-based devices with potential to be commercialized due to its cost-effectiveness, easy fabrication and robust nature of flow control enabling to achieve a viable point-of-care diagnostic platform.

Sequential Reagent Release from a Layered Tablet for Multistep Diagnostic Assays.

Diagnostic assays are commonly performed in multiple steps, where reagents are added at specific times and concentrations into a reaction chamber. The reagents require storage, preparation, and addition in the correct sequence and amount. These steps rely on trained technicians and instrumentation to perform each task. The reliance on such resources hinders the use of these diagnostic assays by lay users. We developed a tablet that can sequentially introduce prequantified lyophilized diagnostic reagents at specific time points for a multistep assay. We designed the tablet to have multiple layers using cellulose-grade polymers, such as microcrystalline cellulose and hydroxypropyl cellulose. Our formulation allows each layer to dissolve at a controlled rate to introduce reagents into the solution sequentially. The release rate is controlled by modulating the compression force or chemical formulation of the layer. Controlling the reagent release time is important because different assays have specific times when reagents need to be added. As proof of concept, we demonstrated two different assays with our tablet system. Our tablet detected nucleic acid target (tpp47 gene from Treponema pallidum) and nitrite ions in an aqueous sample without user intervention. Our multilayer tablets can simplify multistep assay processes.

Microfluidic Paper-based Device for Medicinal Diagnosis.

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. 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. 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. 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.

Application of the aqueous two-phase system and nanozyme signal enhancement for the improved detection of Plasmodium lactate dehydrogenase in serum.

Malaria is an infectious disease that can cause severe sickness and death if not diagnosed and treated in a timely manner. The current gold standard technique for malaria diagnosis is microscopy, which requires a dedicated laboratory setting and trained personnel and can have a long time to result. These requirements can be alleviated using paper-based diagnostic devices that enable rapid and inexpensive diagnosis at the point of care, which can allow patients to receive treatment before their symptoms progress when used for early detection of diseases. The lateral-flow immunoassay (LFA) is one such device, but currently available LFAs are susceptible to false negative results caused by low parasite density. To improve sensitivity and detection, we utilized the aqueous two-phase system (ATPS) to concentrate and purify the sample, and nanozyme signal enhancement to increase the intensity of the visible signal on the test strip. We were able to achieve a limit of detection (LOD) of 0.01ng/mL for the malaria biomarker Plasmodium lactate dehydrogenase (pLDH) in human serum using a multi-step assay combining the LFA format with the ATPS and nanozyme signal enhancement.

In Vitro Mechanisms of Resistance Development to Imipenem-Relebactam in KPC-Producing Klebsiella pneumoniae.

Carbapenem-resistant Enterobacterales, such as KPC-producing Klebsiella pneumoniae, represent a major threat to public health. Novel drug combinations including imipenem-relebactam (IPM-REL) have recently been introduced and have been shown to exhibit excellent activity toward such strains. However, there has recently been reports of the in vivo emergence of IPM-REL resistance in KPC-producing K. pneumoniae. Here, we evaluated, in vitro, the nature of the mutations that lead to IPM-REL resistance in 5 KPC-producing K. pneumoniae strains, including 2 that produce KPC enzymes conferring ceftazidime-avibactam resistance. An in vitro multi-step selection assay was performed and corresponding mutants obtained. Mutations were identified in OmpK36 as well as 2 different mutant derivatives of KPC. Mutant strains exhibited decreased susceptibility to β-lactams, including the carbapenems, and meropenem-vaborbactam (MEM-VAB). Expression of blaKPC gene variants in an Escherichia coli recombinant strain resulted in a concomitant increased susceptibility to carbapenems and decreased susceptibility to CAZ-AVI, and enzymatic assays showed that the inhibitory activity of both AVI and REL was significantly lowered for both KPC mutants compared to parental enzymes. Complementation assays showed that OmpK36 plays a major role in IPM-REL resistance as well resistance to other ß-lactams and β-lactam/ß-lactamase inhibitor combinations. Overall, this study showed that (i) IPM-REL resistant strains can be obtained from CAZ-AVI-susceptible or -resistant KPC producers, (ii) selection of IPM-REL resistance has a collateral effect on MEM-VAB susceptibility - indicative of shared resistance mechanisms, (iii) and mutations in the KPC sequence may be obtained using IPM-REL selection leading to the possibility of vertical and horizontal transfer of this resistance trait.

A perspective on ToF-SIMS analysis of biosensor interfaces: Controlling and optimizing multi-molecular composition, immobilization through bioprinting, molecular orientation

The biosensor interface is the heart of biosensing devices and point-of-need platforms. Analyte detection relies on biosensor surface modifications with different molecules (specific binding molecules, blocking molecules, etc.), accomplished following multi-step functionalization and assay procedures, but discrimination of these molecules is not provided by biosensor response. Mass fabrication of biosensors integrated on a chip is facilitated by spatially-selective functionalization through printing of probe molecules, but the resulting multi-molecular distributions are not readily available. Assay efficiency is determined by the orientation and conformation of probe molecules on biosensor surface, but they are rarely determined during assay development. Above challenges in biosensor functionalization and assay can be solved by microanalysis and chemical imaging provided by Time-of-Flight Secondary Ion Mass Spectrometry. This perspective article shows that biosensor surface modifications can be compared with ToF-SIMS not only to determine but also to optimize multi-molecular composition, bioprinting-based immobilization and molecular orientation. These features are controlled through relevant surface phenomena of adsorption and desorption or droplet evaporation as revealed by ToF-SIMS data. The paper highlights the advantages of molecular discrimination with ToF-SIMS, such as accurate characterization of molecular composition that cannot be deduced only by biosensor response, verification of applied biofunctionalization protocol efficiency, and assay optimization with respect to probe immobilization conditions. Finally, application of ToF-SIMS imaging to biomolecules printed into microarrays, biosensors’ arrays or separate biosensor’s components on a chip are presented, with the printed domains comparable, wider or narrower than the nominal diameter of the printing pin. We hope that the reviewed experimental developments could be an inspiration for future research as to how TOF-SIMS analysis methods could assist in our understanding of complex phenomena taking place onto a surface.

The Deubiquitylase USP5 Knockdown Reduces Semliki Forest Virus Replication in HeLa Cells

Semliki Forest Virus (SFV), a member of the Alphavirus genus in the Togaviridae family, is a small-enveloped, positive-sense single-stranded RNA (+ssRNA) virus. The virus is spread by mosquitos and can infect humans, resulting in mild febrile disease with symptoms that include fever, myalgia, arthralgia, persistent headaches and asthenia. Virulent strains of SFV in mice cause lethal encephalitis by infecting neurons in the central nervous system. In on-going experiments in the research group using a focused siRNA screen we have investigated the role of deubiquitylases (DUBs) during SFV infection (as a model alphavirus) and monitored the effect of DUB depletion on cell viability after infection. We identified a group of DUBs that have a pro-viral effect. The DUB, USP5, from this screen was validated to determine its effect upon viral replication. Here, we show that depleted USP5 in HeLa cells resulted in SFV RNA and viral yield at 8 h post-infection being significantly reduced. In the multi-step viral growth curve assay, in the absence of USP5, similar yields of SFV were determined at 2 and 4 h post-infection. However, a significant reduction in the infectious viral particles release at 6, 8, 10 and 12 h post-infection was observed and this could be reversed by direct constraining viral replication. These results raise the potential for USP5 to play a distinct role in the replication of SFV, suggesting that USP5 may be a possible anti-viral therapy for alphavirus infection.

Pumpless three-dimensional photo paper-based microfluidic analytical device for automatic detection of thioredoxin-1 using enzyme-linked immunosorbent assay.

Microfluidic-based biosensors have been developed for their precise automatic reaction control. However, these biosensors require external devices that are difficult to transport and use. To overcome this disadvantage, our group made an easy-to-use, cheap, and light pumpless three-dimensional photo paper-based microfluidic analytical device (3D-μPAD; weight: 1.5g). Unlike conventional paper-based microfluidic analytical devices, the 3D-μPAD can be used to control fluid flow in a 3D manner, thus allowing sophisticated multi-step reaction control. This device can control fluid flow speed and direction accurately using only the capillary-driven flow without an external device like a pump. The flow speed is controlled by the width of the microfluidic channel and its surface property. In addition, fluid speed control and 3D-bridge structure enable the control of fluid flow direction. Using these methods, multi-step enzyme-linked immunosorbent assay (ELISA) can be done automatically in sequence by injecting solutions (sample, washing, and enzyme's substrate) at the same time in the 3D-μPAD. All the steps can be performed in 14min, and data can be analyzed immediately. To test this device, thioredoxin-1 (Trx-1), a biomarker of breast cancer, is used as the target. In the 3D-μPAD, it can detect 0-200ng/mL of Trx-1, and the prepared 3D-μPAD Trx-1 sensor displays excellent selectivity. Moreover, by analyzing the concentration of Trx-1 in real patients and healthy individuals' blood serum samples using the 3D-μPAD, and comparing results to ELISA, it can be confirmed that the 3D-μPAD is a good tool for cancer diagnosis.

3D Printing of Monolithic Capillarity-Driven Microfluidic Devices for Diagnostics.

Rapid diagnostic testing at the site of the patient is essential when a fully equipped laboratory is not accessible. To maximize the impact of this approach, low-cost, disposable tests that require minimal user-interference and external equipment are desired. Fluid transport by capillary wicking removes the need for bulky ancillary equipment to actuate and control fluid flow. Nevertheless, current microfluidic paper-based analytical devices based on this principle struggle with the implementation of multistep diagnostic protocols because of fabrication-related issues. Here, 3D-printed microfluidic devices are demonstrated in a proof-of-concept enzyme-linked immunosorbent assay in which a multistep assay timeline is completed by precisely engineering capillary wetting within printed porous bodies. 3D printing provides a scalable route to low-cost microfluidic devices and obviates the assembly of discrete components. The resulting rapid and seamless transition between digital data and physical objects allows for rapid design iterations, and opens up perspectives on distributed manufacturing.

Paper Microfluidic Device with a Horizontal Motion Valve and a Localized Delay for Automatic Control of a Multistep Assay.

A microfluidic paper-based analytical device (μPAD) is a cost-effective platform to implement assays, especially for point-of-care testing. Developing μPADs with fluidic control is important to implement multistep assays and provide high sensitivities. However, current localized delays in μPADs made of sucrose have a limited ability to decrease the flow rate. In addition, existing μPADs for automatic multistep assays are limited by their need for auxiliary instruments, their false activation, or their unavoidable tradeoff between available fluid volumes and temporal differences between steps. Here, a novel μPAD composed of a localized dissolvable delay and a horizontal motion mechanical valve for use as an automatic multistep assay is reported. A mixture of fructose and sucrose was used in the localized dissolvable delay and it provided an effective decrease in the flow rate to ensure adequate sensitivity in an assay. The dissolvable delay effectively doubled the flow time. A mechanical valve using a horizontal movement was developed to automatically implement a multistep process. Two-step and four-step processes were enabled with the μPAD. Cardiac troponin I (cTnI), a gold-standard biomarker for myocardial infarction, was used as a model analyte to show the performance of the developed μPAD in an assay. The designed μPAD, with the simple-to-make localized dissolvable delay and the robust mechanical valve, provides the potential to automatically implement high-performance multistep assays toward a versatile platform for point-of-care diagnostics.

Programmable microfluidic flow for automatic multistep digital assay in a single-sheet 3-dimensional paper-based microfluidic device

A single-sheet 3-dimensional paper-based microfluidic analytical device (3D-μPAD) has a user-friendly compact format that eliminates any potential for user alignment error compared to the stacking and folding format. However, achieving sophisticated analysis is still a challenge because forming a complex 3D microfluidic network sufficient for performing analysis in a single sheet of paper is difficult. Here, we present the first report of automatic multistep digital analysis in a single-sheet 3D-μPAD created by controlling the penetration of different wax inks into paper. Simple printing of different wax inks with different spreading properties produces different dimension microchannels in paper. This controllable change in the dimensions of the microchannels results in different flow rates of fluid in each microchannel. Based on the spatial resolution of the microchannels, our strategy enables programming of microfluidic flow through manipulation of the fluidic time delay. This approach creates 3D-μPADs capable of sequential fluid delivery and multistep biochemical reactions with single loading of a sample solution. The methodology can be further expanded to combination with digital assays to measure the concentration of a target analyte by simply counting the number of colored bars at a fixed time. It does not require any external instruments to analyze the measurement of the target analyte concentration. The significance of this work lies in the promising potential for automatic multistep digital assays in a low-cost and easy-to-use paper-based format that can be integrated with a roll-to-roll process for commercial-scale manufacturing.

Using airflow-driven, evaporative gradients to improve sensitivity and fluid control in colorimetric paper-based assays.

Microfluidic paper-based analytical devices (μPADs) are foundational devices for point-of-care testing, yet suffer from limitations in regards to their sensitivity and capability in handling complex assays. Here, we demonstrate an airflow-based, evaporative method that is capable of manipulating fluid flows within paper membranes to offer new functionalities for multistep delivery of reagents and improve the sensitivity of μPADs by 100-1000 times. This method applies an air-jet to a pre-wetted membrane, generating an evaporative gradient such that any solutes become enriched underneath the air-jet spot. By controlling the lateral position of this spot, the solutes in the paper strip are enriched and follow the air jet trajectory, driving the reactions and enhancing visualization for colorimetric readout in multistep assays. The technique has been successfully applied to drive the sequential delivery in multistep immunoassays as well as improve sensitivity for colorimetric detection assays for nucleic acids and proteins via loop-mediated isothermal amplification (LAMP) and ELISA. For colorimetric LAMP detection of the COVID-19 genome, enrichment of the solution on paper could enhance the contrast of the dye in order to more clearly distinguish between the positive and negative results to achieve a sensitivity of 3 copies of SARS-Cov-2 RNAs. For ELISA, enrichment of the oxidized TMB substrate yielded a sensitivity increase of two-to-three orders of magnitude when compared to non-enriched samples - having a limit of detection of around 200 fM for IgG. Therefore, this enrichment method represents a simple process that can be easily integrated into existing detection assays for controlling fluid flows and improving detection of biomarkers on paper.

Nanotechnological immunoassay for rapid label-free analysis of candidate malaria vaccines.

Malaria is a life-threatening epidemic disease with half of the world's population at risk. Although its incidence rate has fallen since 2010, this ratio dramatically stalled between 2014 and 2018. New fast and optimized tools in vaccine analysis and seroconversion testing are critically needed. We developed a clinical diagnostic device based on piezo-actuated nanoresonators that perform as quantitative in situ calibrated nano-bio sensors for specific detection of multiple target molecules in serum samples. The immunoassay successfully diagnoses humoral immune responses induced by malaria vaccine candidates and reveals the timeline and stage of the infection. We applied the newly developed strategy to a variety of different samples, from pure antibody/vaccine solutions, to blood samples from clinical trials on both naïve and pre-exposed malaria volunteers from sub-Saharan countries. Our nanomechanical assay provides a direct one-step label-free quantitative immunoassay that is on par with the gold-standard, multi-step enzyme-linked immunosorbent assay (ELISA). We achieve a limit of detection of few pg ml-1, or sub-pM concentrations. The 6 μl sample volume allows more than 50 experiments from one finger prick. Furthermore, we simultaneously detected multiple analytes by differential functionalization of multiple sensors in parallel. The inherent differential read-out with in situ controls reduces false positive results. Due to the faster turnaround time, the minimal volume required and the automatized handling system, this technique has great potential for miniaturization and routine diagnostics in pandemic emergencies.