This work was supported by the Special Funds for Fundamental Research Funds of institutions of higher-learning affiliated with central departments. We appreciate the support of Classical Prescription Basic Research Team of the Beijing University of Chinese Medicine.

All of the procedures performed in this study were approved by the Ethics Review Committee at the Beijing University of Chinese Medicine (approval number 2017BZYYL00120).
1. Preparation and Characterization of Colloidal Gold
NOTE: For colloidal gold synthesis, as colloidal gold is easily adsorbed on the inner wall of the vessel and is prone to precipitation by impurities, the vessel for synthesis and storage of colloidal gold should be thoroughly cleaned and soaked in acid (40 mL of distilled water, 360 mL of concentrated sulfuric acid, 20 g of potassium dichromate) or subjected to surface passivation treatment. A citric acid reduction method was used to synthesize colloidal gold.
<ol>
	<li>Turn on the magnetic stirrer and place the flask (250 mL) on the mixer.</li>
	<li>Prepare 4% gold chloride acid solution and 1% sodium citrate solution, respectively.</li>
	<li>Add 120 mL of distilled water to a round bottom flask and heat it to boiling on a thermostatic magnetic stirrer.</li>
	<li>Keep boiling, and quickly add 0.5 mL of 4% chloroauric acid and 5 mL of 1% sodium citrate.</li>
	<li>Observe the color of the solution. The pale yellow chloroauric acid solution turns wine red within a few minutes.</li>
	<li>Continue heating for 10 minutes, until the solution changes from colorless to transparent wine red.</li>
	<li>Turn off the power of the thermostatic magnetic mixer, cool to room temperature, and move the mixture to a clean bottle. Store it at 4 &#176;C.</li>
	<li>Determine the size and morphology of the AuNPs by ultraviolet spectroscopy and TEM imaging. 
	&#8203;NOTE: Different sizes of colloid gold particles for various applications can be obtained by changing the proportion of citrate sodium and chloroauric acid.</li>
</ol>
2. Synthesis of AuNPs-mAb Conjugate
NOTE: Since antibodies bind to colloidal gold by electrostatic adsorption, charges on the surface of proteins and colloidal gold directly affect the binding intensity; therefore, the buffer pH value is an important factor affecting the stability of the antibody-colloidal gold conjugate. SSD and anti-SSD mAbs are used as examples in this protocol.
<ol>
	<li>Evaluation of coupling pH
	<ol>
		<li>Add 100 &#956;L of NaCl solution (10% m/v) into eight tubes.</li>
		<li>Adjust the AuNP solutions at pH 5, 6, 7, 8, 9, 10, 11 and 12 with 0.1 M K2CO3.</li>
		<li>Add 100 &#956;L of colloidal gold solution (pH adjusted 5-12) to eight tubes containing NaCl.</li>
		<li>Allow the solutions to stand for several minutes after blending. Observe the color change of each tube solution, and record the tube that remains red.</li>
		<li>Choose the pH value with the least addition of K2CO3 and stable solution color as the optimum pH value for preparing AuNP-mAb conjugates. 
		&#8203;NOTE: Do not use a pH meter because the probe may be destroyed by AuNPs.</li>
	</ol>
	</li>
	<li>Evaluation of antibody amount
	<ol>
		<li>Add 100 &#956;L of NaCl solution (10% m/v) to eight tubes.</li>
		<li>Add 100 &#956;L of colloidal gold solution with optimum pH to each tube.</li>
		<li>Add the monoclonal antibody solutions (protein concentration 0.1 mg/mL-3.2 mg/mL) to the abovementioned eight tubes.</li>
		<li>Allow the solutions to stand for several minutes after blending. Observe the color change of each tube solution, and record the tube that remains red.</li>
		<li>Choose the antibody amount with the lowest concentration of mAb and stable solution color as the optimum mAb amount for preparing AuNP-mAb conjugates.</li>
	</ol>
	</li>
	<li>Prepare the resuspension buffer: add 1 M Tris-HCl (pH 8.8), 1% (w/v) BSA, 0.5% (v/v) Tween 20 and 1% (v/v) PEG 20000 and blend well.</li>
	<li>Synthesis of AuNP-mAb conjugate
	<ol>
		<li>Take 10 mL of colloidal gold solution and use 0.1 M K2CO3 to adjust the solution to the optimal pH value.</li>
		<li>Slowly add SSD mAbs at appropriate concentrations and shake at room temperature for 30 min.</li>
		<li>Centrifuge the mixture at 83 x g (1,000 rpm) for 10 min at 4 &#176;C. Remove the precipitate, which contains impurities or precipitated colloidal gold.</li>
		<li>Centrifuge the mixture at 8,330 x g (10,000 rpm) for 30 min at 4 &#176;C. Discard the supernatant, and the precipitate is the colloidal gold-mAb conjugate.</li>
		<li>Add the resuspension buffer to dissociate the precipitation.</li>
	</ol>
	</li>
</ol>
3. Assembly of the Strip
NOTE: For later flow immunoassays, the selection and pretreatment of membrane material will directly affect the test, which should be investigated. The immunochromatographic strip consists of a sample pad, a conjugate pad, a nitrocellulose (NC) membrane, an absorbent pad and a PVC board (Figure 1). The membrane material should be checked and evaluated by stereomicroscopy to eliminate inhomogeneity.
<ol>
	<li>Paste the NC membrane on a PVC board 2 cm apart from the edge of the suction end of the board.</li>
	<li>Add SSD-BSA (2 mg/mL) dropwise to the NC membrane (2 cm apart from the upper edge) as a test line (1 mm wide), and add goat anti-mouse IgG (1.5 mg/mL) dropwise on the NC membrane (2 cm apart from the lower edge) as a control line (1 mm wide). Control the amount of protein added.</li>
	<li>Attach the absorbent pad to the PVC sheet above the NC membrane and overlap it with the NC membrane by 2 mm.</li>
	<li>Submerge the glass fiber membrane into the AuNPs-mAb conjugate solution. Dry the wet membrane in an incubator at 37 &#176;C.</li>
	<li>Trim the glass fiber membrane to 5 cm long and 2 cm wide and use it as a conjugate pad.</li>
	<li>Paste the pretreated conjugate pad under the NC membrane. The length of overlap with the NC membrane should be 0.1 cm. 
	NOTE: The glass fiber membrane has a strong ability to bind and release proteins.</li>
	<li>Trim the fusion 3 membrane to 1.8 cm long and 3.5 cm wide and use it as the sample pad.</li>
	<li>Paste the sample pad on the PVC board and overlap it with the conjugate pad by 2 mm.</li>
	<li>Cut the assembled paper board into 3.5-mm-wide strips using a cutting machine and compact it using a batch lamination system.</li>
	<li>Finally, place the test strips into the shell, seal them in an aluminum foil bag containing desiccant, and store them away from light. The ICSs are now assembled. 
	&#8203;NOTE: The above is the laboratory procedure. In production, gold spraying equipment and cross-membrane instruments are used to spray gold and make the T and C lines.</li>
</ol>
4. Quantitative Detection
<ol>
	<li>Drop 50 &#181;L of sample solution onto the sample hole to observe the chromatography process. 
	NOTE: As a result of the capillary action driven by the absorbent pad, the sample solution migrates to the other end of the strip. When the sample solution reaches the conjugate pad, the SSD (antigen) in the sample reacts with the AuNPs-mAb preloaded on the pad. When the solution migrates and reaches the T-line, the AuNPs-mAb without SSD can be selectively captured by SSD-BSA (antigen-carrier protein conjugate), showing as a red color on the T-line. Then, the solution migrates to the C line, where the AuNPs-mAb are captured by goat anti-mouse IgG in the region, thus showing as a red color on the C line.</li>
	<li>Analyze the strips with a portable strip reader. The machine can provide the ratio of the test line to the control line (T/C).</li>
	<li>Evaluate the specificity, sensitivity, repeatability and stability of the ICS test. 
	NOTE: Under qualitative detection, one red line indicates a positive result (control line). Two red lines indicate a negative result (test and control lines). If the control line is not present, the test is considered invalid.</li>
</ol>

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The authors have nothing to disclose.

In this work, we present a protocol for the preparation of mAbs against natural product-derived small molecules. The essential steps and the matters needing attention in the procedure have been outlined, and we have demonstrated the utility of this protocol using the small molecule SSD as an example. Example spectra, TEM images, quantitative results and methodological investigations are shown in representative data. Hence, we have demonstrated that the colloidal gold production, AuNP-mAb conjugation and strip assembly st...

Membrane-based lateral flow immunochromatographic strips (ICSs) are useful tools for low-cost and rapid detection. The nitrocellulose membrane as the carrier and colloidal gold as markers of immune chromatography rapid diagnostic reagents are the most commonly used POCT (point of care testing) method, and the testing scope of the project is wider. From their original application in monitoring during pregnancy, their use has been extended to monitor blood coagulation state1,2, myocardial injury3, veterinary medicine4, pesticide residues5, infectious diseases6 and drug concentrations. More types of samples can be assessed, including urine, saliva, whole blood, serum and other body fluids7,8,9.
In recent years, numerous novel assays have been developed for detecting biomarkers in the diagnosis of disorders, including HPLC, UPLC, LC-MS and ELISA, which are sensitive and accurate, credible and specific. However, these methods require sophisticated instrumentation, complex preprocessing and time-consuming treatments9. Hence, developing a more rapid and convenient point-of-care diagnostic strategy for the self- and real-time detection of medicinal active compounds is urgent10,11.
The popularity of ICSs, especially for common tests, is driven by their ease of use, as they do not require professionals or elaborate instrumental setups12. In other words, people who do not have special training can operate strips or self-tests13. The results of the test can be obtained in 5 minutes, which means it can be used for site inspections14. Moreover, according to our calculations, the cost of strips could be lower than 1 RMB15, which means that the tests are inexpensive to promote16. Hence, the ICS is a relatively accurate, simple, and inexpensive disposable device. ICSs based on colloidal gold17,18 are also applied in rapid COVID-19 detection.
The principle of ICS can be divided into sandwich ICS and competitive ICS. Figure 1A is a schematic diagram of the sandwich ICS, which is mainly used for detecting macromolecular substances such as proteins, including tumor markers, inflammatory factors, and human chorionic gonadotropin (HCG, early pregnancy antigen). In this method, paired antibodies targeted at different epitopes of the antigen are used, and the capture antibody is dried on the NC membrane as a test line. Labeled antibody is dried on the conjugate pad, and secondary antibody is used as the control line.
Figure 1B is a schematic diagram of competitive ICS, which is mainly used to detect small molecule substances (MWCO &lt; 2000 Da). The coating antigen is fixed on the NC membrane as a test line, and the labeled antibody is dried on the conjugate pad. During detection, the sample and labeled antibody flow through the detection line under capillary action, and the coated antigen competitively binds free antigen in the sample and develops a red color on the detection line.
Recently, we described the procedure of monoclonal antibody generation against natural products19. In this work, we developed a novel lateral flow immunoassay based on the prepared anti-SSD mA20 for rapid, on-site detection. The results indicate that the immunochromatography assay is an indispensable and convenient tool for detecting natural product-derived compounds.
<img alt="Lateral flow immunoassay diagram; antibody interaction; test and control lines; sample flow path." class="xfigimg" src="/files/ftp_upload/62754/62754fig01.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1 Schematic diagram of the immunochromatography assay (A) Sandwich immunochromatographic test strips. (B) Indirect competitive immune chromatographic test strips. This figure has been modified from Zhang et al., 201821. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62754/62754fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>Chloroauric acid solution (HAuCl&lt;sub&gt;4&lt;/sub&gt;)</td><td>Tianjin Fu Chen Chemical Reagents Factory</td><td>JY-SJ102</td><td></td></tr><tr><td>bovine serum albumin</td><td>AMRESCO</td><td>332</td><td></td></tr><tr><td>centrifuge tube 15 mL</td><td>Corning</td><td>430645</td><td></td></tr><tr><td>centrifuge tube 50 mL</td><td>Corning</td><td>430828</td><td></td></tr><tr><td>ELISA plates, 96 well</td><td>NUNC</td><td>655101</td><td></td></tr><tr><td>Filter paper</td><td>Sinopharm</td><td>H5072</td><td></td></tr><tr><td>Glass fibre membranes</td><td>Jieyi</td><td>XQ-Y6</td><td></td></tr><tr><td>goat-anti-mouse IgG antibody</td><td>applygen</td><td>C1308</td><td></td></tr><tr><td>Nitrocellulose membranes</td><td>Millipore</td><td>millipore 180</td><td></td></tr><tr><td>ovalbumin</td><td>Beijing BIODEE</td><td>5008-25g</td><td></td></tr><tr><td>PEG20000</td><td>Sigma Aldrich</td><td>RNBC6325</td><td></td></tr><tr><td>Pipette 10mL</td><td>COSTAR</td><td>4488</td><td></td></tr><tr><td>Pipette 25mL</td><td>FALCON</td><td>357525</td><td></td></tr><tr><td>semi-rigid PVC sheets</td><td>Jieyi</td><td>JY-C104</td><td></td></tr><tr><td>Sodium citrate</td><td>Beijing Chemical Works</td><td>C1034</td><td></td></tr><tr><td>sodium periodate</td><td>Sinopharm Chemical</td><td>BW-G0008</td><td></td></tr><tr><td>Sulfo-GMBS</td><td>Perbio Science Germany</td><td>22324</td><td></td></tr><tr><td>TipOne Tips 1,000 &amp;micro;L</td><td>Starlab</td><td>S1111-2021</td><td></td></tr><tr><td>Tris-HCl</td><td>Solarbio</td><td>77-86-1</td><td></td></tr><tr><td>TWEEN 20</td><td>Solarbio</td><td>9005-64-5</td><td></td></tr></tbody></table>

development of a lateral flow immunochromatographic strip for rapid and quantitative detection of small molecule compounds

Membrane-based lateral flow immunochromatographic strips (ICSs) are useful tools for low-cost self-diagnosis and have been efficiently applied to toxin, physiological index and clinical biomarker detection. In this protocol, we provide a detailed description of the steps to develop a rapid, sensitive and quantitative lateral-flow immunoassay (using AuNPs as a marker and mAbs as a probe). The procedure describes the preparation and characterization of colloidal gold, synthesis of the AuNP-mAb conjugate, assembly of the immunochromatographic strip, and methodological investigation of the assay. The results showed that the final strips can be further utilized for the rapid and convenient self-diagnosis of a small molecule, which may provide an alternative tool in the rapid and precise analysis of physiological and biological indices.

Characterization of colloidal gold 
The prepared colloidal gold solutions were claret red. TEM analyses were used to determine the morphology and shape of AuNPs (Figure 2A-D). Figure 2A and Figure 2B reveal that the particles are polyhedral in shape and uniformly distributed. The average diameter of AuNPs was found to be approximately 14 nm (Figure 2C). A high-resolu...

Watch this Scientific Journal Video about Development of a Lateral Flow Immunochromatographic Strip for Rapid and Quantitative Detection of Small Molecule Compounds at JoVE.com

Development of a Lateral Flow Immunochromatographic Strip for Rapid and Quantitative Detection of Small Molecule Compounds

Membrane-based lateral flow immunochromatographic strips (ICSs) are useful tools for low-cost self-diagnosis and have been efficiently applied to toxin, ...

development-lateral-flow-immunochromatographic-strip-for-rapid

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

7.8K Views.  Beijing University of Chinese Medicine. De procedure beschrijft de bereiding van colloïdaal goud en de assemblage van de immunochromatografische strips. Ten eerste werden chlooraurinezuur en natriumcitraat gebruikt voor de synthese van colloïdaal goud. Natriumcitraat werd toegevoegd aan chloroaurinezuuroplossing en gekookt om colloïdaal goud met de juiste deeltjesgrootte te verkrijgen.Synthese, het colloïdaal goud antilichaam conjugaat. Na centrifugering werden colloïdale gouddeeltjes opgelost in de juiste oplossing en ...

Video: Development of a Lateral Flow Immunochromatographic Strip for Rapid and Quantitative Detection of Small Molecule Compounds

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Bioengineering

Microfluidic Chip Fabrication and Method to Detect Influenza

Fast and effective diagnostics play an important role in controlling infectious disease by enabling effective patient management and treatment. Here, we present an integrated microfluidic thermoplastic chip with the ability to amplify influenza A virus in patient nasopharyngeal (NP) swabs and aspirates. Upon loading the patient sample, the microfluidic device sequentially carries out on-chip cell lysis, RNA purification and concentration steps within the solid phase extraction (SPE), reverse transcription (RT) and polymerase chain reaction (PCR) in RT-PCR chambers, respectively. End-point detection is performed using an off-chip Bioanalyzer (Agilent Technologies, Santa Clara, CA). For peripherals, we used a single syringe pump to drive reagent and samples, while two thin film heaters were used as the heat sources for the RT and PCR chambers. The chip is designed to be single layer and suitable for high throughput manufacturing to reduce the fabrication time and cost. The microfluidic chip provides a platform to analyze a wide variety of virus and bacteria, limited only by changes in reagent design needed to detect new pathogens of interest.

An integrated microfluidic thermoplastic chip has been developed for use as a molecular diagnostic. The chip performs nucleic acid extraction, reverse transcriptase, and PCR. Methods for fabricating and running the chip are described.

Fast  and effective diagnostics play an important role in controlling infectious  disease by enabling effective patient management and treatment. Here, we ...

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

Fabricating Cotton Analytical Devices

A robust, low-cost analytical device should be user-friendly, rapid, and affordable. Such devices should also be able to operate with scarce samples and provide information for follow-up treatment. Here, we demonstrate the development of a cotton-based urinalysis (i.e., nitrite, total protein, and urobilinogen assays) analytical device that employs a lateral flow-based format, and is inexpensive, easily fabricated, rapid, and can be used to conduct multiple tests without cross-contamination worries. Cotton is composed of cellulose fibers with natural absorptive properties that can be leveraged for flow-based analysis. The simple but elegant fabrication process of our cotton-based analytical device is described in this study. The arrangement of the cotton structure and test pad takes advantage of the hydrophobicity and absorptive strength of each material. Because of these physical characteristics, colorimetric results can persistently adhere to the test pad. This device enables physicians to receive clinical information in a timely manner and shows great potential as a tool for early intervention.

To investigate simple fabrication approaches for multiple assay needs, we created a fluid-absorbing channel system made of cotton material. This device was used to establish a multiple detection platform, and solve contamination issues that commonly affect lateral flow-based biomedical devices, for clinical urinalysis of nitrite, total protein, and urobilinogen.

A robust, low-cost analytical device should be user-friendly, rapid, and affordable. Such devices should also be able to operate with scarce samples and ...

Computer Numerical Control Micromilling of a Microfluidic Acrylic Device with a Staggered Restriction for Magnetic Nanoparticle-Based Immunoassays

Microfluidic systems have greatly improved immunoassay techniques. However, many microfabrication techniques require specialized, expensive, or complicated equipment, making fabrication costly and incompatible with mass production, which is one of the most important preconditions for point-of-care tests (POCT) to be adopted in low-resource settings. This work describes the fabrication process of an acrylic (polymethylmethacrylate, PMMA) device for nanoparticle-conjugated enzymatic immunoassay testing using the computer numerical control (CNC) micromilling technique. The functioning of the microfluidic device is shown by performing an immunoassay to detect a commercial antibody using lysozyme as a model antigen conjugated to 100 nm magnetic nanoparticles. This device integrates a physical staggered restriction of only 5 &#181;m in height, used to capture magnetic microparticles that make up a magnetic trap by placing an external magnet. In this way, the magnetic force on the immunosupport of conjugated nanoparticles is enough to capture them and resist flow drag. This microfluidic device is particularly suitable for low-cost mass production without the loss of precision for immunoassay performance.

Microfluidics is a powerful tool for the development of diagnostic tests. However, expensive equipment and materials, as well as laborious fabrication and handling techniques, are often required. Here, we detail the fabrication protocol of an acrylic microfluidic device for magnetic micro- and nanoparticle-based immunoassays in a low-cost and simple-to-use setting.

Microfluidic systems have greatly improved immunoassay techniques. However, many microfabrication techniques require specialized, expensive, or ...

Engineering

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

Quantum Dot Nanobeads for Enhanced Sensitivity in Biomarker Detection

Fluorescent Lateral Flow Immunoassay Based on Quantum Dots Nanobeads

Quantum dots, also known as semiconductor nanocrystals, are novel fluorescent labels for biological imaging and sensing. However, quantum dot-antibody conjugates with small dimensions (~10 nm), prepared through laborious purification procedures, exhibit limited sensitivity in detecting certain trace disease markers using lateral flow immunoassay strips. Herein, we present a method for the preparation of quantum dot nanobeads (QDNB) using a one-step emulsion evaporation method. Using the as-prepared QDNB, a fluorescent lateral flow immunoassay was fabricated to detect disease biomarkers using C-reactive protein (CRP) as an example. Unlike single quantum dot nanoparticles, quantum dot nanobead-antibody conjugates are more sensitive as immunoassay labels due to signal amplification by encapsulating hundreds of quantum dots in one polymer composite nanobead. Moreover, the larger size of QDNBs facilitates easier centrifugation separation when conjugating QDNBs with antibodies. The fluorescent lateral flow immunoassay based on QDNBs was fabricated, and the CRP concentration in the sample was measured in 15 min. The test results can be qualitatively assessed under UV light illumination and quantitatively measured using a fluorescent reader within 15 min.

Author Spotlight: High-Quality Quantum Dot Nanobeads for Sensitive Fluorescent Lateral Flow Immunoassays

Here, we describe a protocol for the preparation of quantum dot nanobeads (QDNB) and the detection of disease biomarkers using QDNB-based lateral flow immunoassay strips. The test results can be qualitatively assessed under UV light illumination and quantitatively measured using a fluorescent strip reader within 15 min.

Quantum dots, also known as semiconductor nanocrystals, are novel fluorescent labels for biological imaging and sensing. However, quantum dot-antibody ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

Immunology and Infection

CRISPR-Cas Synthetic Urine Biomarker Test for Cancer Diagnostics

CRISPR-Cas-mediated Multianalyte Synthetic Urine Biomarker Test for Portable Diagnostics

Creating synthetic biomarkers for the development of precision diagnostics has enabled detection of disease through pathways beyond those used for traditional biofluid measurements. Synthetic biomarkers generally make use of reporters that provide readable signals in the biofluid to reflect the biochemical alterations in the local disease microenvironment during disease incidence and progression. The pharmacokinetic concentration of the reporters and biochemical amplification of the disease signal are paramount to achieving high sensitivity and specificity in a diagnostic test. Here, a cancer diagnostic platform is built using one format of synthetic biomarkers: activity-based nanosensors carrying chemically stabilized DNA reporters that can be liberated by aberrant proteolytic signatures in the tumor microenvironment. Synthetic DNA as a disease reporter affords multiplexing capability through its use as a barcode, allowing for the readout of multiple proteolytic signatures at once. DNA reporters released into the urine are detected using CRISPR nucleases via hybridization with CRISPR RNAs, which in turn produce a fluorescent or colorimetric signal upon enzyme activation. In this protocol, DNA-barcoded, activity-based nanosensors are constructed and their application is exemplified in a preclinical mouse model of metastatic colorectal cancer. This system is highly modifiable according to disease biology and generates multiple disease signals simultaneously, affording a comprehensive understanding of the disease characteristics through a minimally invasive process requiring only nanosensor administration, urine collection, and a paper test which enables point-of-care diagnostics.

Author Spotlight: Engineering Molecular Tools for Disease Detection and Imaging

This protocol describes a CRISPR-Cas-mediated, multianalyte synthetic urine biomarker test that enables point-of-care cancer diagnostics through the ex vivo analysis of tumor-associated protease activities.

Creating synthetic biomarkers for the development of precision diagnostics has enabled detection of disease through pathways beyond those used for ...

Smartphone-Integrated Dengue NS1 Paper-Based Diagnostic Device

Portable Paper-Based Immunoassay Combined with Smartphone Application for Colorimetric and Quantitative Detection of Dengue NS1 Antigen

Dengue virus (DENV) infection, which is transmitted by Aedes mosquitoes, is a major public health concern in tropical and subtropical countries. With an annual incidence of approximately 10 million cases and 20,000-25,000 deaths, particularly among children, there is an urgent need for practical diagnostic tools. The presence of dengue non-structural protein 1 (NS1) during early infection has been linked to cytokine release, vascular leakage, and endothelial dysfunction, making it a potential marker for severe dengue. 
Paper-based immunoassays such as lateral flow assays (LFAs) and microfluidic paper-based analytical devices (PADs) have gained popularity as diagnostic tests due to their simplicity, rapidity, inexpensiveness, specificity, and ease of interpretation. However, conventional paper-based immunoassays for dengue NS1 detection typically rely on visual inspection, yielding only qualitative results. To address this limitation and enhance sensitivity, we proposed a highly portable NS1 dengue detection assay on a Paper-based Analytical Device (PAD), namely, DEN-NS1-PAD, that integrates a smartphone application as a colorimetric and quantitative reader. The development system enables direct quantification of NS1 concentrations in clinical samples. 
Serum and blood samples obtained from patients were utilized to demonstrate the system prototype performance. The results were obtained immediately and can be employed for clinical assessment, both in well-equipped healthcare facilities and resource-limited settings. This innovative combination of a paper-based immunoassay with a smartphone application offers a promising approach for enhanced detection and quantification of dengue NS1 antigen. By augmenting sensitivity beyond the capabilities of the naked eye, this system holds great potential for improving clinical decision-making in dengue management, particularly in remote or underserved areas.

Author Spotlight: Development of a Smartphone-Enhanced Paper-Based Device for Rapid Dengue NS1 Detection

Addressing urgent dengue diagnostic needs, here we introduce a smartphone app-integrated Dengue NS1 Paper-based Analytical Device (DEN-NS1-PAD) for quantifying Dengue NS1 antigen concentration in clinical serum/blood samples. This innovation enhances dengue management by aiding clinical decision-making in various healthcare settings, even resource-limited ones.

Dengue virus (DENV) infection, which is transmitted by Aedes mosquitoes, is a major public health concern in tropical and subtropical countries. With an ...

Development of a Lateral Flow Immunochromatographic Strip for Rapid and Quantitative Detection of Small Molecule Compounds

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