This work was supported by the Project of Shanghai Science and Technology Committee (STCSM) (22S31902000) and the Clinical Research Incubation Program of Shanghai Skin Disease Hospital (NO. lcfy2021-10).

The study was approved by the Institutional Review Board of Shanghai Skin Disease Hospital (No. 2020-15). All experimental procedures involving human blood samples were conducted in a Biosafety Level II laboratory. The details of the reagents and equipment used in this study are listed in the Table of Materials.
1. Preparation of QDs nanobeads
NOTE: For QD nanobead synthesis, an emulsion-solvent evaporation technique was used to synth...

Quantum Dot Nanobeads for Enhanced Sensitivity in Biomarker Detection

<ol>
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Here, we describe a protocol for the preparation of quantum dot nanobeads (QDNB)27 and the use of QDNB for the preparation of fluorescent lateral flow immunoassays (LFIA). The qualitative and quantitative measurement of CRP in samples is demonstrated. This QDNB-based LFIA can also be applied to other disease biomarkers25,32, food toxins29,30, viruses16,...

Lateral flow immunoassay (LFIA) strips serve as crucial rapid detection tools at point-of-care1,2, particularly in disease screening during epidemics. However, traditional colloidal gold-based LFIA test strips exhibit low detection sensitivity and only provide qualitative results3. To enhance the detection sensitivity of LFIA, various new nanoparticles have emerged, including colored latex4,5, upconversion fluorescent nanoparticles6, time-resolved fluorescent microspheres7,8, and quantum dots9,10,11. Quantum dots (QDs)12,13, also known as semiconductor nanocrystals, offer tunable emission wavelengths, a wide excitation range, and high luminescence efficiency, making them ideal labels for biological imaging.
However, the fluorescence signal emitted by individual quantum dots remains weak, resulting in relatively low detection sensitivity in immunoassays. Encapsulation of numerous quantum dots within microspheres can amplify signals and improve the sensitivity of quantum dot-based immunoassays. Various methods, such as layer-by-layer self-assembly14,15,16,17,18, the swelling method19,20, and silica microsphere21,22,23,24 encapsulation, have been employed to encapsulate quantum dots inside microspheres. For example, quantum dot-functionalized silica nanosphere labels can be achieved by increasing QD loading per sandwiched immunoreaction25. A spray dryer equipped with an ultrasonic atomizer has also been used to prepare nanoscale QD-BSA nanospheres26. However, the aforementioned methods suffer from complex multi-steps, fluorescence quenching, and low productivity.
In our previous work27, an emulsion-solvent evaporation method for encapsulating quantum dots inside polymer nanobeads was reported. This preparation technique is simple, maintains the fluorescent efficiency of QDs, ensures high encapsulation efficiency, and allows for easy scalable production. Several research groups have successfully developed LFIA strips using QDNBs prepared through this method for applications, including food toxin detection28,29,30, infectious disease biomarker detection31,32, and environmental monitoring33.
This protocol presents specific preparation steps for quantum dot nanobeads (QDNB), QDNB and antibody conjugation, preparation of QDNB-based LFIA, and measurement of C-reactive protein (CRP) in human plasma samples.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>(dimethylamino)propyl)-N&#8217;-ethylcarbodiimide hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>03450</td>
			<td></td>
		</tr>
		<tr>
			<td>Absorbance paper&#160;</td>
			<td>Kinbio Biotech</td>
			<td>CH37K</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>B2064</td>
			<td></td>
		</tr>
		<tr>
			<td>Casein</td>
			<td>Sigma-Aldrich</td>
			<td>C8654</td>
			<td></td>
		</tr>
		<tr>
			<td>CdSe/ZnS quantum dot</td>
			<td>Suzhou Mesolight Inc.</td>
			<td>CdSe/ZnS-625</td>
			<td></td>
		</tr>
		<tr>
			<td>Choloroform</td>
			<td>Sino Pharm</td>
			<td>10006818</td>
			<td></td>
		</tr>
		<tr>
			<td>CRP antibody</td>
			<td>Hytest Biotech</td>
			<td>4C28</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent lateral flow assay reader</td>
			<td>Suzhou Helmence Precision Instrument</td>
			<td>FIC-H1</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass fiber pad</td>
			<td>Kinbio Biotech</td>
			<td>SB06</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG</td>
			<td>Sangon Biotech</td>
			<td>D111018</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose membrane&#160;</td>
			<td>Satorious</td>
			<td>CN140</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(styrene-maleic anhydride) copolymer&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S458066</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit IgG</td>
			<td>Sangon Biotech</td>
			<td>D110502</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate</td>
			<td>Sino Pharm</td>
			<td>30166428</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Sino Pharm</td>
			<td>10019718</td>
			<td></td>
		</tr>
	</tbody>
</table>

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

Fluorescent Lateral Flow Immunoassay Based on Quantum Dots Nanobeads

Our research interests currently focus on constructing high-quality multifunctional nanomaterials and their application in the development of ultrasensitive point-of-care diagnostics. Lateral flow immunoassay strips serve as critical rapid detection tools at the point-of-care, particularly in disease screening during epidemics to enhance the detection sensitivity to lateral flow immunoassay. Various new nanoparticle has emerged, including colored latex, upcoercion fluorescent nanoparticles, timed-result fluorescent microspheres, and quantum dots.We presented a one-step emulsion evaporation method for preparing quantum dot nanobeads and other applications in sensitivity, for instance, lateral fluoro-immunoassays. Common to other methods of preparing quantum dot nanobeads, this protocol offers the advantage of simplicities of operation and high productivity. Additionally, the polymer composite nanobeads are biocompatible, unlike silica nanobeads, which generally need additional surface modification with polymers.Sensitive fluorescent lateral flow immunoassay strips have been developed using prepared quantum dot nanobeads. These strips can be used to qualitatively and quantitatively detect disease biomarkers at point-of-care. This new assay could help medical professionals diagnose diseases quickly and accurately.

The QDNB preparation procedures are schematically illustrated in Figure 1A. The oil phase containing QDs and polymer in chloroform was mixed with the water phase, and a mini-emulsion was obtained by sonication. The emulsion was solidified by gradual evaporation of chloroform. The transmission electron micrograph (TEM) of QDNB is presented in Figure 2A. The QDNBs have a spherical morphology, with average diameters of 96 nm, measured over 50 QDNBs in TEM images. Q...

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 ...

fluorescent-lateral-flow-immunoassay-based-on-quantum-dots-nanobeads

Research

JoVE Journal

Biochemistry

379 ビュー.  Tongji University. 現在、私たちの研究対象は、高品質の多機能ナノ材料の構築と、超高感度ポイントオブケア診断の開発におけるそれらの応用に焦点を当てています。ラテラルフローイムノアッセイストリップは、特に流行時の疾患スクリーニングにおいて、ポイントオブケアにおける重要な迅速検出ツールとして機能し、ラテラルフローイムノアッセイに対す?...

ビデオ: 著者スポットライト:高感度蛍光ラテラルフローイムノアッセイのための高品質量子ドットナノビーズ

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...

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preparation of quantum dots (qds) nanobeads for qdnb-based lateral flow immunoassay

Preparation of Quantum Dots (QDs) Nanobeads for QDNB-Based Lateral Flow Immunoassay

nanofabrication of gate-defined gaas/algaas lateral quantum dots

Nanofabrication of Gate-defined GaAs/AlGaAs Lateral Quantum Dots

This paper presents a detailed fabrication protocol for gate-defined semiconductor lateral quantum dots on gallium arsenide heterostructures. These nanoscale devices are used to trap few electrons for use as quantum bits in quantum information processing or for other mesoscopic experiments such as coherent conductance...

correlative light- and electron microscopy using quantum dot nanoparticles

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of epoxy embedded human pathology tissue. We employ commercial antibody fragment conjugated QDs that are visualized by widefield fluorescence light microscopy and transmission electron...

resonance fluorescence of an ingaas quantum dot in a planar cavity using orthogonal excitation and detection

Resonance Fluorescence of an InGaAs Quantum Dot in a Planar Cavity Using Orthogonal Excitation and Detection

Resonant excitation of a single self-assembled quantum dot can be achieved using an excitation mode orthogonal to the fluorescence collection mode. We demonstrate a method using the waveguide and Fabry-Perot modes of a planar microcavity surrounding the quantum dots. The method allows complete freedom in the detection...

sensitive measurement of mitophagy by flow cytometry using the ph-dependent fluorescent reporter mt-keima

Sensitive Measurement of Mitophagy by Flow Cytometry Using the pH-dependent Fluorescent Reporter mt-Keima

Mitophagy, the selective degradation of mitochondria, has been implicated in mitochondrial homeostasis and is deregulated in various human diseases. However, convenient experimental methods for measuring mitophagy activity are very limited. Here, we provide a sensitive assay for measuring mitophagy activity using flow...

high-throughput confocal imaging of quantum dot-conjugated sars-cov-2 spike trimers to track binding and endocytosis in hek293t cells

High-throughput Confocal Imaging of Quantum Dot-Conjugated SARS-CoV-2 Spike Trimers to Track Binding and Endocytosis in HEK293T Cells

In this protocol, quantum dots conjugated to recombinant SARS-CoV-2 spike enable cell-based assays to monitor spike binding to hACE2 at the plasma membrane and subsequent endocytosis of the bound proteins into the...

acquisition of high-quality digital video of drosophila larval and adult behaviors from a lateral perspective

Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective

Here we describe a simple and widely accessible microscopy technique to acquire high-quality digital video of Drosophila adult and larval mutant phenotypes from a lateral...

Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective

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

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, ...

enhanced electron injection and exciton confinement for pure blue quantum-dot light-emitting diodes by introducing partially oxidized aluminum cathode

Enhanced Electron Injection and Exciton Confinement for Pure Blue Quantum-Dot Light-Emitting Diodes by Introducing Partially Oxidized Aluminum Cathode

A protocol is presented for fabricating high-performance, pure blue ZnCdS/ZnS-based quantum dots light-emitting diodes by employing an autoxidized aluminum...

simultaneous detection of different antibody classes in a multiplexed serological test

Author Spotlight: Expanding the Scope of Multiplex Immunoassays for Lyme Borreliosis Diagnostics and Pathogen Research 

A three-channel dual-reporter fluorescence flow analysis system was used to develop a bead-based multiplex immunoassay that simultaneously evaluates serum samples for IgG and IgM elicited against multiple antigens of different Borrelia species that cause Lyme borreliosis in Europe and North...

Multiplex Immunoassay for &lt;em&gt;Borrelia&lt;/em&gt;-Specific IgG and IgM in Serum

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real-time imaging of axonal transport of quantum dot-labeled bdnf in primary neurons

Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons

Axonal transport of BDNF, a neurotrophic factor, is critical for the survival and function of several neuronal populations. Some degenerative disorders are marked by disruption of axonal structure and function.  We demonstrated the techniques used to examine live trafficking of QD-BDNF in microfluidic chambers using primary neurons.

wide-field fluorescent microscopy and fluorescent imaging flow cytometry on a cell-phone

Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone

We review our recent results on the integration of fluorescent microscopy and imaging flow cytometry tools on a cell-phone using compact and cost-effective opto-fluidic attachments. These cell-phone based micro-analysis devices might be useful for cytometric analysis, such as performing various cell counting tasks as well as for high-throughput screening of e.g., water samples in resource limited...

quality-controlled sputum analysis by flow cytometry

Quality-Controlled Sputum Analysis by Flow Cytometry

This protocol describes an efficient method for dissociating sputum into a single cell suspension and the subsequent characterization of cellular subsets on standard flow cytometric platforms.

fabrication of silica ultra high quality factor microresonators

Fabrication of Silica Ultra High Quality Factor Microresonators

We describe the use of a carbon dioxide laser reflow technique to fabricate silica resonant cavities, including free-standing microspheres and on-chip microtoroids.  The reflow method removes surface imperfections, allowing long photon lifetimes within both devices.  The resulting devices have ultra high quality factors, enabling applications ranging from telecommunications to...

fabrication of three-dimensional paper-based microfluidic devices for immunoassays

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based...

measuring membrane lipid turnover with the ph-sensitive fluorescent lipid analog nd6

Measuring Membrane Lipid Turnover with the pH-sensitive Fluorescent Lipid Analog ND6

This protocol presents a fluorescence imaging method that uses a class of pH-sensitive lipid fluorophores to monitor lipid membrane trafficking during cell exocytosis and the endocytosis...

preparation of high-quality fermented fish product

Preparation of High-Quality Fermented Fish Product

The goal of the protocol is to provide an industrialized fish fermentation technique based on inoculation of Saccharomyces...

rapid assessment of membrane protein quality by fluorescent size exclusion chromatography

Rapid Assessment of Membrane Protein Quality by Fluorescent Size Exclusion Chromatography

The present protocol describes a procedure to perform fluorescent size exclusion chromatography (FSEC) on membrane proteins to assess their quality for downstream functional and structural analysis. Representative FSEC results collected for several G-protein coupled receptors (GPCRs) under detergent-solubilized and detergent-free conditions are...

To begin, mix 0.4 milliliters of polystyrene co-maleic anhydride solution and 0.1 milliliters of QD solution to prepare 1 milliliter of the oil phase. Then fill the rest with chloroform until the volume reaches 1 milliliter. Next, prepare the aqueous phase using sodium dodecyl sulfate solution.Combine 1 milliliter of the oil phase and 5 milliliters of the aqueous phase in a vial. Place the vial on a magnetic stir and turn it on. Once the oil and aqueous phases are thoroughly mixed, immediately transfer the vial to the programmed ultrasonication device and turn on the power.Then place the vial back on the magnetic stir at room temperature and stir continuously overnight. After solvent evaporation, add 0.1 milliliters of 0.1 millimolar sodium hydroxide to the nanobead suspension, and allow it to react for one hour.

QDNB-Antibody Conjugates Preparation Using One-Step Emulsion Evaporation Method

To begin, add 100 microliters of prepared QD nanobeads to 200 microliters of phosphate buffer. Then add six microliters of EDC. After that, incubate the mixture for 30 minutes at room temperature on a rotary mixer.Centrifuge the mixture at 13, 523 g for 10 minutes at room temperature and discard the supernatant. Next, add 100 microliters of phosphate buffer to the precipitate. Cover the tube with its lid and immerse it in the operational ultrasonic water bath until the precipitate is fully dispersed.Disperse 100 micrograms of antibody in 200 microliters of phosphate buffer, and add the activated QD nanobead suspension to the antibody solution. Incubate the mixture at room temperature for 30 minutes with continuous rotation. Then remove excess antibodies by centrifugation at 6, 010 g for five minutes at room temperature, and carefully discard the supernatant.To block the nanobeads, add 200 microliters of 1%casein solution to the residue. Cover the tube with its lid and immerse it into the operational ultrasonic water bath until the residue is fully dispersed. Incubate the mixture at room temperature for two hours with continuous rotation.Add 100 microliters of preservative solution to the mixture and store at four degrees Celsius for subsequent use.

Lateral Flow Immunoassay Strips Preparation, Assay Operation and its Quantitative Evaluation

To begin, prepare the test line, and control line coating solution. Dilute the C-reactive protein capture antibody, and Goat anti-rabbit immunoglobulin G with phosphate buffer. Next, place the polyvinyl chloride board properly and remove the protective paper located 25 millimeters from the edge of the board.Align and adhere the nitrocellulose membrane along the lower edge of the remaining protective paper to the polyvinyl chloride board. Dispense the test line, and control line coating solutions onto the nitrocellulose membrane, ensuring it is placed 20 millimeters apart from the upper edge, forming a test line. For drying, place the nitrocellulose membrane in conditions with a humidity of less than 30%and a temperature range of 18 to 26 degrees Celsius.To prepare the conjugate pad, submerge the conjugate pad into the conjugate pad treatment solution, then place the soaked conjugate pad in a drying oven for overnight drying treatment. Dilute the prepared QD nano bead antibody conjugate solution with the preservative solution, and dispense it onto the conjugate pad. For sample pad preparation.Submerge the sample pad into the sample pad treatment solution. Then place the soaked sample pad into a drying oven set at 45 degrees Celsius, and dry for 24 hours. Successively, place the absorbent paper, conjugate pad and sample pad on the polyvinyl chloride board with the nitrocellulose membrane.Pipette 10 microliters of plasma sample or calibrator solution into one milliliter of A PBS buffer containing 0.1%tween 20. Then transfer 100 microliters of the prepared diluted sample onto the sample pad of the test strip. After a 15 minute run, observe the fluorescent signals of the test line and control line under ultraviolet light exposure to obtain qualitative results.Insert the strip card into a fluorescent lateral flow assay reader to obtain the fluorescence intensities for quantitative analysis.