Name: fluorescent lateral flow immunoassay based on quantum dots nanobeads
Uploaded: 2024-06-28T14:40:00.000+00:00
Duration: 7 min 13 s
Description: Quantum dots, also known as semiconductor nanocrystals, are novel fluorescent labels for biological imaging and sensing. However, quantum dot-antibody ...

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

<ol>
	<li>de Puig, H., Bosch, I., Gehrke, L., Hamad-Schifferli, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+of+the+nano-bio+interface+in+lateral+flow+and+dipstick+immunoassays.">Challenges of the nano-bio interface in lateral flow and dipstick immunoassays.</a> Trends Biotechnol. 35 (12), 1169-1180 (2017).</li><li>Miller, B. S., 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=Spin-enhanced+nanodiamond+biosensing+for+ultrasensitive+diagnostics.">Spin-enhanced nanodiamond biosensing for ultrasensitive diagnostics.</a> Nature. 587 (7835), 588-593 (2020).</li><li>Gao, Z., 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=Platinum-decorated+gold+nanoparticles+with+dual+functionalities+for+ultrasensitive+colorimetric+in+vitro+diagnostics.">Platinum-decorated gold nanoparticles with dual functionalities for ultrasensitive colorimetric in vitro diagnostics.</a> Nano Lett. 17 (9), 5572-5579 (2017).</li><li>Fan, L., 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=Deeply-dyed+nanobead+system+for+rapid+lateral+flow+assay+testing+of+drugs+at+point-of-care.">Deeply-dyed nanobead system for rapid lateral flow assay testing of drugs at point-of-care.</a> Sensors Actuators B Chem. 362, 131829 (2022).</li><li>Garcia, V. S., Guerrero, S. A., Gugliotta, L. M., Gonzalez, V. D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+lateral+flow+immunoassay+based+on+colored+latex+particles+for+detection+of+canine+visceral+leishmaniasis.">A lateral flow immunoassay based on colored latex particles for detection of canine visceral leishmaniasis.</a> Acta Trop. 212, 105643 (2020).</li><li>You, M., 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=Household+fluorescent+lateral+flow+strip+platform+for+sensitive+and+quantitative+prognosis+of+heart+failure+using+dual-color+upconversion+nanoparticles.">Household fluorescent lateral flow strip platform for sensitive and quantitative prognosis of heart failure using dual-color upconversion nanoparticles.</a> ACS Nano. 11 (6), 6261-6270 (2017).</li><li>Ye, Z., Tan, M., Wang, G., Yuan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+fluorescent+europium+chelate-doped+silica+nanoparticles:+preparation,+characterization+and+time-resolved+fluorometric+application.">Novel fluorescent europium chelate-doped silica nanoparticles: preparation, characterization and time-resolved fluorometric application.</a> J Mater Chem. 14 (5), 851 (2004).</li><li>Xu, Y., Li, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+fluorescent+labeling+of+silica+nanoparticles+with+lanthanide+chelates+for+highly+sensitive+time-resolved+immunofluorometric+assays.">Multiple fluorescent labeling of silica nanoparticles with lanthanide chelates for highly sensitive time-resolved immunofluorometric assays.</a> Clin Chem. 53 (8), 1503-1510 (2007).</li><li>Zhang, B., 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=Improving+detection+sensitivity+by+oriented+bioconjugation+of+antibodies+to+quantum+dots+with+a+flexible+spacer+arm+for+immunoassay.">Improving detection sensitivity by oriented bioconjugation of antibodies to quantum dots with a flexible spacer arm for immunoassay.</a> RSC Adv. 6 (55), 50119-50127 (2016).</li><li>Li, Z., 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+sensitive+detection+of+protein+biomarker+using+a+portable+fluorescence+biosensor+based+on+quantum+dots+and+a+lateral+flow+test+strip.">Rapid and sensitive detection of protein biomarker using a portable fluorescence biosensor based on quantum dots and a lateral flow test strip.</a> Anal Chem. 82 (16), 7008-7014 (2010).</li><li>Wang, L., 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=Fluorescent+strip+sensor+for+rapid+determination+of+toxins.">Fluorescent strip sensor for rapid determination of toxins.</a> Chem Commun. 47 (5), 1574-1576 (2011).</li><li>Medintz, I. L., Uyeda, H. T., Goldman, E. R., Mattoussi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantum+dot+bioconjugates+for+imaging,+labeling+and+sensing.">Quantum dot bioconjugates for imaging, labeling and sensing.</a> Nat Mater. 4 (6), 435-446 (2005).</li><li>Smith, A. M., Nie, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compact+quantum+dots+for+single-molecule+imaging.">Compact quantum dots for single-molecule imaging.</a> J Vis Exp. (68), e4236 (2012).</li><li>Wang, C., 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=Layer-by-layer+assembly+of+magnetic-core+dual+quantum+dot-shell+nanocomposites+for+fluorescence+lateral+flow+detection+of+bacteria.">Layer-by-layer assembly of magnetic-core dual quantum dot-shell nanocomposites for fluorescence lateral flow detection of bacteria.</a> Nanoscale. 12 (2), 795-807 (2020).</li><li>Hu, J., Tang, F., Jiang, Y. Z., Liu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+screening+and+quantitative+detection+of+Salmonella+using+a+quantum+dot+nanobead-based+biosensor.">Rapid screening and quantitative detection of Salmonella using a quantum dot nanobead-based biosensor.</a> Analyst. 145 (6), 2184-2190 (2020).</li><li>Wang, W., 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=Introduction+of+graphene+oxide-supported+multilayer-quantum+dots+nanofilm+into+multiplex+lateral+flow+immunoassay:+A+rapid+and+ultrasensitive+point-of-care+testing+technique+for+multiple+respiratory+viruses.">Introduction of graphene oxide-supported multilayer-quantum dots nanofilm into multiplex lateral flow immunoassay: A rapid and ultrasensitive point-of-care testing technique for multiple respiratory viruses.</a> Nano Res. 16 (2), 3063-3073 (2023).</li><li>Wang, C., 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=Colorimetric-fluorescent+dual-signal+enhancement+immunochromatographic+assay+based+on+molybdenum+disulfide-supported+quantum+dot+nanosheets+for+the+point-of-care+testing+of+monkeypox+virus.">Colorimetric-fluorescent dual-signal enhancement immunochromatographic assay based on molybdenum disulfide-supported quantum dot nanosheets for the point-of-care testing of monkeypox virus.</a> Chem Eng J. 472, 144889 (2023).</li><li>Zheng, S., 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=Dual-color+MoS2@QD+nanosheets+mediated+dual-mode+lateral+flow+immunoassay+for+flexible+and+ultrasensitive+detection+of+multiple+drug+residues.">Dual-color MoS2@QD nanosheets mediated dual-mode lateral flow immunoassay for flexible and ultrasensitive detection of multiple drug residues.</a> Sensors Actuators B Chem. 403, 135142 (2024).</li><li>Wang, G., 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=Efficient+incorporation+of+quantum+dots+into+porous+microspheres+through+a+solvent-evaporation+approach.">Efficient incorporation of quantum dots into porous microspheres through a solvent-evaporation approach.</a> Langmuir. 28 (14), 6141-6150 (2012).</li><li>Li, H., 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=Fluorescent+lateral+flow+immunoassay+for+highly+sensitive+detection+of+eight+anticoagulant+rodenticides+based+on+cadmium-free+quantum+dot-encapsulated+nanospheres.">Fluorescent lateral flow immunoassay for highly sensitive detection of eight anticoagulant rodenticides based on cadmium-free quantum dot-encapsulated nanospheres.</a> Sensors Actuators B Chem. 324, 128771 (2020).</li><li>Gao, F., 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=Rational+design+of+dendritic+mesoporous+silica+nanoparticles'+surface+chemistry+for+quantum+dot+enrichment+and+an+ultrasensitive+lateral+flow+immunoassay.">Rational design of dendritic mesoporous silica nanoparticles' surface chemistry for quantum dot enrichment and an ultrasensitive lateral flow immunoassay.</a> ACS Appl Mater Interfaces. 13 (18), 21507-21515 (2021).</li><li>Xu, L. D., Zhu, J., Ding, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoassay+of+SARS-CoV-2+nucleocapsid+proteins+using+novel+red+emission-enhanced+carbon+dot-based+silica+spheres.">Immunoassay of SARS-CoV-2 nucleocapsid proteins using novel red emission-enhanced carbon dot-based silica spheres.</a> Analyst. 146 (16), 5055-5060 (2021).</li><li>Tao, S., 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=SARS-Cov-2+Spike-S1+antigen+test+strip+with+high+sensitivity+endowed+by+high-affinity+antibodies+and+brightly+fluorescent+QDs/silica+nanospheres.">SARS-Cov-2 Spike-S1 antigen test strip with high sensitivity endowed by high-affinity antibodies and brightly fluorescent QDs/silica nanospheres.</a> ACS Appl Mater Interfaces. 15 (23), 27612-27623 (2023).</li><li>Wang, C., 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=Development+of+an+ultrasensitive+fluorescent+immunochromatographic+assay+based+on+multilayer+quantum+dot+nanobead+for+simultaneous+detection+of+SARS-CoV-2+antigen+and+influenza+A+virus.">Development of an ultrasensitive fluorescent immunochromatographic assay based on multilayer quantum dot nanobead for simultaneous detection of SARS-CoV-2 antigen and influenza A virus.</a> Sensors Actuators B Chem. 345, 130372 (2021).</li><li>Chen, L., Chen, C., Li, R., Li, Y., Liu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CdTe+quantum+dot+functionalized+silica+nanosphere+labels+for+ultrasensitive+detection+of+biomarker.">CdTe quantum dot functionalized silica nanosphere labels for ultrasensitive detection of biomarker.</a> Chem Commun. 19, 2670-2672 (2009).</li><li>Chu, M., 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=A+novel+method+for+preparing+quantum+dot+nanospheres+with+narrow+size+distribution.">A novel method for preparing quantum dot nanospheres with narrow size distribution.</a> Nanoscale. 2 (4), 542-547 (2010).</li><li>Zhang, P., Lu, H., Chen, J., Han, H., Ma, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+and+sensitive+detection+of+HBsAg+by+using+a+quantum+dots+nanobeads+based+dot-blot+immunoassay.">Simple and sensitive detection of HBsAg by using a quantum dots nanobeads based dot-blot immunoassay.</a> Theranostics. 4 (3), 307-315 (2014).</li><li>Ouyang, S., 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=An+on-site,+ultra-sensitive,+quantitative+sensing+method+for+the+determination+of+total+aflatoxin+in+peanut+and+rice+based+on+quantum+dot+nanobeads+strip.">An on-site, ultra-sensitive, quantitative sensing method for the determination of total aflatoxin in peanut and rice based on quantum dot nanobeads strip.</a> Toxins. 9 (4), 137 (2017).</li><li>Liu, J., 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=Quantitative+ciprofloxacin+on-site+rapid+detections+using+quantum+dot+microsphere+based+immunochromatographic+test+strips.">Quantitative ciprofloxacin on-site rapid detections using quantum dot microsphere based immunochromatographic test strips.</a> Food Chem. 335, 127596 (2021).</li><li>Chen, Y., Fu, Q., Xie, J., Wang, H., Tang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+high+sensitivity+quantum+dot-based+fluorescent+quenching+lateral+flow+assay+for+the+detection+of+zearalenone.">Development of a high sensitivity quantum dot-based fluorescent quenching lateral flow assay for the detection of zearalenone.</a> Anal Bioanal Chem. 411 (10), 2169-2175 (2019).</li><li>Zhang, Q., 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=SARS-CoV-2+detection+using+quantum+dot+fluorescence+immunochromatography+combined+with+isothermal+amplification+and+CRISPR/Cas13a.">SARS-CoV-2 detection using quantum dot fluorescence immunochromatography combined with isothermal amplification and CRISPR/Cas13a.</a> Biosens Bioelectron. 202, 113978 (2022).</li><li>Zhong, X., 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=CRISPR-based+quantum+dot+nanobead+lateral+flow+assay+for+facile+detection+of+varicella-zoster+virus.">CRISPR-based quantum dot nanobead lateral flow assay for facile detection of varicella-zoster virus.</a> Appl Microbiol Biotechnol. 107 (10), 3319-3328 (2023).</li><li>Liu, Y., Xiao, M., Xu, N., Yang, M., Yi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Point-of-need+quantitation+of+2,4-dichlorophenoxyacetic+acid+using+a+ratiometric+fluorescent+nanoprobe+and+a+smartphone-based+sensing+system.">Point-of-need quantitation of 2,4-dichlorophenoxyacetic acid using a ratiometric fluorescent nanoprobe and a smartphone-based sensing system.</a> Sensors Actuators B Chem. 367, 132083 (2022).</li><li>McCafferty, C., 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=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" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+and+quantitative+detection+of+C-reaction+protein+based+on+immunofluorescent+nanospheres+coupled+with+lateral+flow+test+strip.">Sensitive and quantitative detection of C-reaction protein based on immunofluorescent nanospheres coupled with lateral flow test strip.</a> Anal Chem. 88 (12), 6577-6584 (2016).</li><li>Fan, L., 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=One-component+dual-readout+aggregation-induced+emission+nanobeads+for+qualitative+and+quantitative+detection+of+c-reactive+protein+at+the+point+of+care.">One-component dual-readout aggregation-induced emission nanobeads for qualitative and quantitative detection of c-reactive protein at the point of care.</a> Anal Chem. 96 (1), 401-408 (2024).</li><li>Gui, Y., 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=Colorimetric+and+reverse+fluorescence+dual-signal+readout+immunochromatographic+assay+for+the+sensitive+determination+of+sibutramine.">Colorimetric and reverse fluorescence dual-signal readout immunochromatographic assay for the sensitive determination of sibutramine.</a> ACS Omega. 9 (6), 7075-7084 (2024).</li></ol>

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><tbody><tr><td>(dimethylamino)propyl)-N&amp;rsquo;-ethylcarbodiimide hydrochloride</td><td>Sigma-Aldrich</td><td>03450</td><td></td></tr><tr><td>Absorbance paper&amp;nbsp;</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&amp;nbsp;</td><td>Satorious</td><td>CN140</td><td></td></tr><tr><td>Poly(styrene-maleic anhydride) copolymer&amp;nbsp;</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.

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...

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.

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 ...

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

Research

JoVE Journal

Biochemistry

1.1K Views.  Tongji University. Onze onderzoeksinteresses richten zich momenteel op het construeren van hoogwaardige multifunctionele nanomaterialen en hun toepassing in de ontwikkeling van ultragevoelige point-of-care diagnostiek. Laterale flow-immunoassaystrips dienen als kritische snelle detectie-instrumenten op het zorgpunt, met name bij ziektescreening tijdens epidemieën om de detectiegevoeligheid voor laterale flow-immunoassay te verbeteren. Er zijn verschillende nieuwe nanodeeltjes ...

Video: Auteur in de schijnwerpers: hoogwaardige Quantum Dot nanobeads voor gevoelige fluorescerende laterale flow-immunoassays

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

Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different downstream patient-to-patient transmission potentials, and require very different patient treatments. Thus, recent Zika outbreaks make it urgent to develop tools that rapidly discriminate these viruses in patients and trapped mosquitoes, to select the correct patient treatment, and to understand and manage their epidemiology in real time.
Unfortunately, current diagnostic tests, including those receiving 2016 emergency use authorizations and fast-track status, detect viral RNA by reverse transcription polymerase chain reaction (RT-PCR), which requires instrumentation, trained users, and considerable sample preparation. Thus, they must be sent to &#34;approved&#34; reference laboratories, requiring time. Indeed, in August 2016, the Center for Disease Control (CDC) was asking pregnant women who had been bitten by a mosquito and developed a Zika-indicating rash to wait an unacceptable 2 to 4 weeks before learning whether they were infected. We very much need tests that can be done on site, with few resources, and by trained but not necessarily licensed personnel.
This video demonstrates an assay that meets these specifications, working with urine or serum (for patients) or crushed mosquito carcasses (for environmental surveillance), all without much sample preparation. Mosquito carcasses are captured on paper carrying quaternary ammonium groups (Q-paper) followed by ammonia treatment to manage biohazards. These are then directly, without RNA isolation, put into assay tubes containing freeze-dried reagents that need no chain of refrigeration. A modified form of reverse transcription loop-mediated isothermal amplification with target-specific fluorescently tagged displaceable probes produces readout, in 30 min, as a three-color fluorescence signal. This is visualized with a handheld, battery-powered device with an orange filter. Forward contamination is prevented with sealed tubes, and the use of thermolabile uracil DNA glycosylase (UDG) in the presence of dUTP in the amplification mixture.

Current multiplexed diagnostics to detect Zika, chikungunya, and dengue viruses require complex sample preparation and expensive instrumentation, and are difficult to use in low resource environments. We show a diagnostic that uses isothermal amplification with target-specific strand displaceable probes to detect and differentiate these viruses with high sensitivity and specificity.

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

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.

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

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 human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.

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 microscopy.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using ...

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

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 ...

Immunology and Infection

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

The development of new technologies for cellular fluorescence microscopy has facilitated high-throughput screening methods for drug discovery. Quantum dots are fluorescent nanoparticles with excellent photophysical properties imbued with bright and stable photoluminescence as well as narrow emission bands. Quantum dots are spherical in shape, and with the proper modification of the surface chemistry, can be used to conjugate biomolecules for cellular applications. These optical properties, combined with the ability to functionalize them with biomolecules, make them an excellent tool for investigating receptor-ligand interactions and cellular trafficking. Here, we present a method that uses quantum dots to track the binding and endocytosis of SARS-CoV-2 spike protein. This protocol can be used as a guide for experimentalists looking to utilize quantum dots to study protein-protein interactions and trafficking in the context of cellular physiology.

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 cytoplasm.

The development of new technologies for cellular fluorescence microscopy has facilitated high-throughput screening methods for drug discovery. Quantum ...

Biology

Multianalyte Immunobead Assay: A Flow-Based Immunoassay Technique for Concurrent Detection of Multiple Antibody Subtypes in Human Serum Sample

Source: Fhied, C. L. et al., <a href="https://www.jove.com/v/63352">Dynamic Monitoring of Seroconversion using a Multianalyte Immunobead Assay for Covid-19.</a> J. Vis. Exp. (2022).
This video describes an immunoassay technique using fluorescent magnetic beads or microspheres coupled with different antigens to simultaneously detect various antibody isotypes in a serum sample. The assay is helpful in understanding seroconversion during an infection or post-vaccination.

Source: Fhied, C. L. et al., Dynamic Monitoring of Seroconversion using a Multianalyte Immunobead Assay for Covid-19. J. Vis. Exp. (2022).
This video ...

JoVE Encyclopedia of Experiments

Biological Techniques

Assay Techniques

Immunoassays

A Magnetic Nanoparticle-Based Immunoassay Using a Microfluidic Device

Source: Hern&#225;ndez-Ortiz, J. A., et al. <a href="https://app.jove.com/v/63899">Computer Numerical Control Micromilling of a Microfluidic Acrylic Device with a Staggered Restriction for Magnetic Nanoparticle-Based Immunoassays.</a> J. Vis. Exp. (2022).
This video demonstrates a microfluidic system for a magnetic nanoparticle-based immunoassay. The detection system utilizes a microfluidic device incorporating a magnetic trap within its channels. Pre-formed immunocomplexes, comprised of antigen-coated magnetic nanoparticles bound to primary and peroxidase-conjugated secondary antibodies, are introduced into the channels. The immunocomplexes become captured in the porous magnetic trap upon applying an external magnetic field. Using a fluorogenic substrate emitting fluorescence upon catalysis by the peroxidases, the captured immunocomplexes are detected under a microscope.

1. Device preparation

	Fill the channels with distilled water using a syringe. Make sure there are no leaks or resistance to flow. Immerse the device in ...

Fluorescent Lateral Flow Immunoassay Based on Quantum Dots Nanobeads

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video