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 synthesize QD nanobeads, with a ratio of oil phase to water phase of 1:5. The mini emulsion is generated via ultrasonication, and the nanobeads are solidified through solvent (chloroform) evaporation. Sodium hydroxide was utilized to catalyze the hydrolysis of anhydride groups on the nanobead surface, converting them into carboxyl groups.
<ol>
	<li>Preparation of oil phase and aqueous phase solutions
	<ol>
		<li>Prepare 1 mL of oil phase: 0.4 mL of poly (styrene-co-maleic anhydride) (PSMA) solution (50 mg/mL), 0.1 mL of QDs solution (100 mg/mL), fill any volume less than 1 mL with chloroform.</li>
		<li>Prepare 5 mL of aqueous phase: SDS aqueous solution (0.5 wt %).</li>
	</ol>
	</li>
	<li>Preparation of emulsion and solidification
	<ol>
		<li>Add 1 mL of oil phase and 5 mL of aqueous phase to a vial (15 mL), and place the vial on a magnetic stirrer. Turn on the power of the magnetic stirrer.</li>
		<li>After the oil and aqueous phases are completely mixed, immediately transfer the flask to the programmed (60 s, a 3 s pulse followed by a 3 s pause, 50% amplitude) ultrasonication and turn on the power.</li>
		<li>Observe the solution after ultrasonication. The initially crimson-colored solution transitioned to an opalescent phase that still retains a reddish undertone.</li>
		<li>Place the vial&#160;back on the magnetic stirrer at room temperature, continuously stirring overnight. The chloroform was gradually evaporated, and the emulsion solidified into composite nanobeads.</li>
	</ol>
	</li>
	<li>Surface modification of nanobeads
	<ol>
		<li>Add 0.1 mL of 0.1 mM sodium hydroxide to the nanobeads suspension for 1 h. The anhydride groups on the surface of nanobeads get hydrolyzed into carboxyl groups.</li>
	</ol>
	</li>
	<li>Purification of nanobeads
	<ol>
		<li>Turn off the magnetic stirrer and move the solidified nanobeads to a centrifuge tube. Centrifuge the tube at 13523 x g for 10 min at room temperature, and discard the supernatant.</li>
		<li>Add the initial volume of deionized water to the precipitation, cover the tube with its lid, submerge it into the ultrasonic water bath, and maintain immersion until the precipitate is fully dispersed.</li>
		<li>Repeat steps 1.4.1-1.4.2 twice, and finally, transfer the nanobeads to the glass bottle. Store it at 4 &#176;C until use.</li>
	</ol>
	</li>
	<li>Characterization of nanobeads
	<ol>
		<li>Characterize the size and morphology of the QDs nanobeads using transmission electronic microscopy (TEM) imaging22. 
		NOTE: Different sizes of nanobeads can be obtained by changing the proportion of QDs solution and PSMA solution. The size of the QDs nanobeads can also be determined through dynamic light scattering (DLS)27.</li>
	</ol>
	</li>
</ol>
2. Preparation of QDNB-antibody conjugates
<ol>
	<li>Activation of carboxyl groups
	<ol>
		<li>Add 100 &#181;L of prepared QDNB (10 mg/mL) in 200 &#181;L of phosphate buffer (PB, 20 mM, pH 6.0), then add 6 &#181;L of EDC (20 mg/mL).</li>
		<li>Incubate the mixture for 30 min at room temperature on a rotary mixer.</li>
		<li>After the incubation, centrifuge the mixture at 13523 x g for 10 min at room temperature and discard the supernatant.</li>
		<li>Add 100 &#181;L of PB (20 mM, pH 6.0) to the precipitation, cover the tube with its lid, and immerse the tube into the operational ultrasonic water bath until the precipitate is fully dispersed.</li>
	</ol>
	</li>
	<li>Conjugation of antibody and nanobeads
	<ol>
		<li>Disperse 100 &#181;g antibody in 200 &#181;L of PB (20 mM, pH 6.0) and add the activated QDNB suspension obtained from step 2.1 into the antibody solution.</li>
		<li>Incubate the mixture at room temperature for 30 min with continuous rotation. Remove excess antibodies by centrifugation at 6010 x g for 5 min (at room temperature), and then carefully discard the supernatant.</li>
	</ol>
	</li>
	<li>Blocking of nanobeads
	<ol>
		<li>Add 200 &#181;L of 1% casein solution (in 20 mM PB buffer, pH 7.4) to the residue (obtained in step 2.2.2), cover the tube with its lid, and immerse it into the operational ultrasonic water bath until the residue is fully dispersed.</li>
		<li>Incubate the mixture at room temperature for 2 h with continuous rotation. 
		NOTE: The objective of this step is to block any unreacted sites on the surface of QDNB.</li>
	</ol>
	</li>
	<li>Storage of conjugates
	<ol>
		<li>Add 100 &#181;L of preservative solution (20 mM PB buffer, pH 7.4, 1% BSA, 5% trehalose, 6% sucrose, 0.1% S9, pH 7.4) to the mixture and store at 4 &#176;C for subsequent use.</li>
	</ol>
	</li>
</ol>
3. Preparation of lateral flow immunoassay strips
NOTE: The immunochromatographic strip is composed of a nitrocellulose (NC) membrane, a sample pad, a conjugate pad, an absorbent pad, and a polyvinyl chloride (PVC) board (Figure 1). All solutions should be prepared and used immediately. It is recommended that lateral flow immunoassay strips be prepared in a clean room with controlled humidity.
<ol>
	<li>Preparation of NC membrane
	<ol>
		<li>Prepare test line (T-line) and control line (C-line) coating solution: dilute the CRP capture antibody and goat anti-rabbit IgG with PB to a final concentration of 1 mg/mL.</li>
		<li>Place the PVC board properly, and remove the protective paper located 25 mm from the edge of the PVC board. Align and adhere the NC membrane along the lower edge of the remaining protective paper to the PVC board.</li>
		<li>Dispense T-line and C-line coating solutions onto the NC membrane at a jetting rate of 1 &#181;L/cm, ensuring it is placed 20 mm apart from the upper edge, forming a test line (1 mm wide).</li>
		<li>Place the NC membrane in conditions with a humidity of less than 30% and a temperature range of 18-26 &#176;C for drying. The drying time should be at least 24 h. 
		NOTE: Ensure the top and bottom edges of the NC membrane are flush with the corresponding edges of the adjacent areas. NC membranes should not be touched with hands. The humidity during this step should be controlled within the range of 45% to 65%. Before proceeding, the T-line coating solution, C-line coating solution, and NC membrane should be equilibrated to the same temperature.</li>
	</ol>
	</li>
	<li>Preparation of the conjugate pad
	<ol>
		<li>Submerge the conjugate pad into the conjugate pad treatment solution (20 mM PB buffer, pH 7.4, 1%BSA).</li>
		<li>Place the soaked conjugate pad into a drying oven for drying treatment (room temperature) overnight.</li>
		<li>Gently adhere the modified conjugate pad onto the PVC board.</li>
		<li>Dilute the prepared QDNB-antibody conjugate solution with the preservative solution (20 mM PB buffer, pH 7.4, 1% BSA, 5% trehalose, 6% sucrose, 0.1% S9, pH 7.4).</li>
		<li>Dispense the QDNB-antibody conjugate solution onto the conjugate pad at a jetting rate of 1 &#181;L/cm and dry the conjugate pad for 24 h. 
		NOTE: The specific dilution ratio should be adjusted according to the detection target.</li>
	</ol>
	</li>
	<li>Preparation of sample pad
	<ol>
		<li>Submerge the sample pad into the sample pad treatment solution (20 mM PB buffer, pH 7.4, 1% BSA, 5% trehalose, 6% sucrose, 0.1% S9, pH 7.4).</li>
		<li>Place the soaked sample pad into a drying oven (45 &#176;C) and dry for 24 h.</li>
	</ol>
	</li>
	<li>Assembling the card and cutting it into strips
	<ol>
		<li>As shown in Figure 1, successively place the absorbent paper (absorbent pad), conjugate pad, and sample pad on the PVC board with NC membrane.</li>
		<li>Cut the card into 3.8 mm width strips.</li>
	</ol>
	</li>
	<li>Packaging of the strips
	<ol>
		<li>Encase the test strips in a plastic cassette and seal an aluminum foil pouch that contains a desiccant.</li>
	</ol>
	</li>
</ol>
4. Assay operation and qualitative evaluation
NOTE: Operations involving human blood samples are recommended to be conducted in a Biosafety Level II laboratory. The human plasma samples were obtained from a clinical laboratory (from deidentified human subjects). Typically, human blood was collected using an EDTA vacuum tube, and plasma was isolated according to the standard protocol34. Centrifuge at 3,000 &#215; g for 10 min at room temperature for plasma separation. Collect the yellow plasma upper layer using a pipette, and store the plasma in a 1.5 ml tube at -80 &#176;C until use.
<ol>
	<li>Pipette 10 &#181;L of plasma sample or calibrator solution into 1 mL of a PBS buffer containing 0.1% Tween 20. Then, transfer 100 &#181;L of the prepared diluted sample onto the sample pad of the test strip.</li>
	<li>After a 15 min run, observe the fluorescence signals of the T line and C line under ultraviolet lightexposure to obtain qualitative results.</li>
	<li>Insert the strip card into a fluorescent lateral flow assay reader to obtain the fluorescence intensities for quantitative analysis. 
	NOTE: In this example, the sandwich immunoassay method is employed35,36; one red line (control line) indicates a negative result, while the two red lines (test and control lines) indicate a positive result. The absence of the control line indicates that the test conducted is invalid.</li>
</ol>

Quantum Dot Nanobeads for Enhanced Sensitivity in Biomarker Detection

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href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+collection+processing+and+handling+for+plasma+and+serum+proteomics+BT+-+Serum/plasma+proteomics.">Blood collection processing and handling for plasma and serum proteomics BT - Serum/plasma proteomics.</a> Methods Mol Biol. 2628, 33-40 (2023).</li><li>Zhang, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+quantitative+detection+of+C-reactive+protein+based+on+quantum+dots+and+immunofiltration+assay.">Rapid and quantitative detection of C-reactive protein based on quantum dots and immunofiltration assay.</a> Int J Nanomedicine. 10, 6161-6173 (2015).</li><li>Hu, J., et al. <a target="_blank" 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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 ...

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

Research

JoVE Journal

Biochemistry

486 Views.  Tongji University. 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, t...