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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S., Guerrero, S. A., Gugliotta, L. M., Gonzalez, V. D. 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Pengfei Zhang is a founder of Shanghai Kundao Biotech and has potential financial interests with this company.

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 ...

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Research

JoVE Journal

Biochemistry

387 Görüntüleme.  Tongji University. Araştırma ilgi alanlarımız şu anda yüksek kaliteli çok işlevli nanomalzemelerin oluşturulmasına ve bunların ultra hassas hasta başı teşhislerinin geliştirilmesinde uygulanmasına odaklanmaktadır. Yanal akış immünoassay şeritleri, yanal akış immünolojik testine karşı algılama duyarlılığını artırmak için özellikle salgınlar sırasında hastalık taramasında, bakım noktasında kritik hızlı tespit araçları olarak hizmet ed...