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" 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, viruses16,27, and more.
The critical steps in this protocol are the preparation of QDNB and QDNB-antibody conjugates. The particle size of QDNB and the QD content in nanobeads should be optimized to obtain highly fluorescent QDNB with low background signals in the LFIA. Capture and detection antibody pairs are critical for the immunoassay and should be screened for the best matches from various vendors. Conjugation of the antibody with QDNB should be optimized, including the antibody/QDNB ratio, buffer pH, and blocking reagents. Specifically, aggregation of QDNBs and antibodies should be avoided by optimizing the pH of the buffer, salt concentration, and reaction time in the conjugation procedures.
Compared with other preparation methods for quantum dot nanobeads, this protocol has the advantages of simplicity, easy operation, and high productivity. Moreover, the polymer composite nanobeads are biocompatible, unlike silica nanobeads, which usually require additional surface modification with polymers. However, the QDNBs prepared using this protocol are not as monodisperse as those prepared using polymer19 or silica21 template nanobeads. The particle size of QDNBs usually ranges from 70 nm to 200 nm.
QDNB-based lateral flow immunoassay (LFIA) strips are prepared similarly to other fluorescent LFIAs. Due to the high quantum yields of QDs and the signal amplification of QDNBs, the QDNB-based LFIA using this protocol is more sensitive than other fluorescent microsphere-based LFIAs, such as time-resolved fluorescent microspheres and fluorescein microspheres. Additionally, the stability of QDNB is superior to traditional fluorescein, providing QDNB-based LFIAs with a longer storage time.
However, cadmium on the surface of quantum dots is prone to chelation with sulfhydryl groups in proteins, especially in aqueous environments. Avoiding the use of high concentrations of protein is critical for the stability of QDNB-based LFIAs. Cadmium is toxic, and its usage is limited in some situations, necessitating the preparation of cadmium-free quantum dots with high quantum yields and stability.
In summary, a versatile method for preparing quantum dot nanobeads in one step is provided, and it is scalable for industrial production. Using the as-prepared QDNBs, fluorescent lateral flow immunoassay strips were created for the qualitative and quantitative detection of disease biomarkers. The ideal QDNB for LFIA is a small-sized nanobead encapsulated with numerous QDs without fluorescence quenching.

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.

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. QD nanoparticles are clearly observed inside the nanobeads, indicating successful encapsulation. Figure 2B and Figure 2D show the fluorescence and absorption spectra of QDNB, respectively.
The QDNB and antibody were conjugated using carbodiimide chemistry, as shown in Figure 1B. The QDNB-antibody conjugate was used to prepare QDNB-based LFIA. The structure of LFIA is illustrated in Figure 1C. If the CRP antigen is present in the sample, the QDNB-anti-CRP conjugates accumulate at the test line.
As shown in Figure 3, the measurement is carried out by adding 100 &#181;L of diluted sample to the sample hole. After a reaction time of 15 min, the test results can be qualitatively read under UV light illumination. The quantitative results can be obtained using a fluorescent strip reader, and the concentration of the sample can be calculated using a calibration equation embedded in the strip reader software37,38.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67000/67000fig01.jpg" /> 
Figure 1: Illustration of the fluorescent lateral flow immunoassay based on quantum dots nanobeads. (A) Procedures for the preparation of QDNB. (B) Principle of QDNB-antibody conjugates. (C) Principle of QDNB-based LFIA for the detection of CRP. <a href="https://www.jove.com/files/ftp_upload/67000/67000fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67000/67000fig02.jpg" /> 
Figure 2: Characterization of QDNBs. (A) TEM image of QDNBs. Scale bar: 100 nm. (B) Fluorescent emission spectra of QDNB. (C) Absorption spectra of QDNB. <a href="https://www.jove.com/files/ftp_upload/67000/67000fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/67000/67000fig03.jpg" /> 
Figure 3: Procedures of lateral-flow immunoassay for CRP. (A) Illustration of the detection. (B) Photographs of QDNB-based LFIA strips detecting CRP samples in a range of 0-30 mg/L under UV light. (C) Fluorescent reader for quantification of results. (D) Photograph of the quantitative results. (E) Detection of C-reactive protein (CRP) using logistic transformation followed by linear regression. <a href="https://www.jove.com/files/ftp_upload/67000/67000fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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998 Views.  Tongji University. 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.

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

Fabricating a UV-Vis and Raman Spectroscopy Immunoassay Platform

Immunoassays are used to detect proteins based on the presence of associated antibodies. Because of their extensive use in research and clinical settings, a large infrastructure of immunoassay instruments and materials can be found. For example, 96- and 384-well polystyrene plates are available commercially and have a standard design to accommodate ultraviolet-visible (UV-Vis) spectroscopy machines from various manufacturers. In addition, a wide variety of immunoglobulins, detection tags, and blocking agents for customized immunoassay designs such as enzyme-linked immunosorbent assays (ELISA) are available.
Despite the existing infrastructure, standard ELISA kits do not meet all research needs, requiring individualized immunoassay development, which can be expensive and time-consuming. For example, ELISA kits have low multiplexing (detection of more than one analyte at a time) capabilities as they usually depend on fluorescence or colorimetric methods for detection. Colorimetric and fluorescent-based analyses have limited multiplexing capabilities due to broad spectral peaks. In contrast, Raman spectroscopy-based methods have a much greater capability for multiplexing due to narrow emission peaks. Another advantage of Raman spectroscopy is that Raman reporters experience significantly less photobleaching than fluorescent tags1. Despite the advantages that Raman reporters have over fluorescent and colorimetric tags, protocols to fabricate Raman-based immunoassays are limited. The purpose of this paper is to provide a protocol to prepare functionalized probes to use in conjunction with polystyrene plates for direct detection of analytes by UV-Vis analysis and Raman spectroscopy. This protocol will allow researchers to take a do-it-yourself approach for future multi-analyte detection while capitalizing on pre-established infrastructure.

Nanoparticle-based optical probes have been designed as a vehicle for detecting antigens using Raman and UV-Vis spectroscopy. Here we describe a protocol for preparing such probes for a UV-Vis/Raman spectroscopy immunoassay in such a way to incorporate future multiplexing capabilities.

Immunoassays are used to detect proteins based on the presence of associated antibodies. Because of their extensive use in research and clinical settings, ...

DNA Staining Method Based on Formazan Precipitation Induced by Blue Light Exposure

DNA staining methods are very important for biomedical research. We designed a simple method that allows DNA visualization to the naked eye by the formation of a colored precipitate. It works by soaking the acrylamide or agarose DNA gel in a solution of 1x (equivalent to 2.0 &#181;M) SYBR Green I (SG I) and 0.20 mM nitro blue tetrazolium that produces a purple precipitate of formazan when exposed to sunlight or specifically blue light. Also, DNA recovery tests were performed using an ampicillin resistant plasmid in an agarose gel stained with our method. A larger number of colonies was obtained with our method than with traditional staining using SG I with ultraviolet illumination. The described method is fast, specific, and non-toxic for DNA detection, allowing visualization of biomolecules to the "naked eye" without a transilluminator, and is inexpensive and appropriate for field use. For these reasons, our new DNA staining method has potential benefits to both research and industry.

This method allows selective staining and quantification of DNA in gels by soaking the gel in a SYBR Green I/Nitro Blue Tetrazolium solution and then exposing it to sunlight or a blue light source. This produces a visible precipitate and requires almost no equipment, making it ideal for field use.

DNA staining methods are very important for biomedical research. We designed a simple method that allows DNA visualization to the naked eye by the ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM c-Cyts) has recently emerged as a novel whole-cell analytical method to characterize the bacterial electron transport from the respiratory chain to the cell exterior, referred to as the extracellular electron transport (EET). While the pathway and kinetics of the electron flow during the EET reaction have been investigated, a whole-cell electrochemical method to examine the impact of cation transport associated with EET has not yet been established. In the present study, an example of a biochemical technique to examine the deuterium kinetic isotope effect (KIE) on EET through OM c-Cyts using a model microbe, Shewanella oneidensis MR-1, is described. The KIE on the EET process can be obtained if the EET through OM c-Cyts acts as the rate-limiting step in the microbial current production. To that end, before the addition of D2O, the supernatant solution was replaced with fresh media containing a sufficient amount of the electron donor to support the rate of upstream metabolic reactions, and to remove the planktonic cells from a uniform monolayer biofilm on the working electrode. Alternative methods to confirm the rate-limiting step in microbial current production as EET through OM c-Cyts are also described. Our technique of a whole-cell electrochemical assay for investigating proton transport kinetics can be applied to other electroactive microbial strains.

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Here we present a protocol of whole-cell electrochemical experiments to study the contribution of proton transport to the rate of extracellular electron transport via the outer-membrane cytochromes complex in Shewanella oneidensis MR-1.

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM ...

Fluorescent Silver Staining of Proteins in Polyacrylamide Gels

Silver staining is a colorimetric technique widely used to visualize protein bands in polyacrylamide gels following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The classic silver stains have certain drawbacks, such as high background staining, poor protein recovery, low reproducibility, a narrow linear dynamic range for quantification, and limited compatibility with mass spectrometry (MS). Now, with the use of a fluorogenic Ag+ probe, TPE-4TA, we developed a fluorescent silver staining method for the total protein visualization in polyacrylamide gels. This new stain avoids the troublesome silver reduction step in traditional silver stains. Moreover, the fluorescent silver stain demonstrates good reproducibility, sensitivity, and linear quantification in protein detection, making it a useful and practical protein gel stain.

Here, we describe a detailed protocol outlining a new fluorescent staining technique for total protein detection in polyacrylamide gels. The protocol utilizes a silver ion-specific fluorescence turn-on probe, which detects Ag+-protein complexes, and eliminates certain limitations of traditional chromogenic silver stains.

Silver staining is a colorimetric technique widely used to visualize protein bands in polyacrylamide gels following sodium dodecyl sulfate-polyacrylamide ...

Imaging of Extracellular Vesicles by Atomic Force Microscopy

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane proteins and other molecules, and diverse luminal content inherited from a parent cell, which includes RNAs, proteins, and DNAs. The characterization of the hydrodynamic sizes of EVs, which depends on the size of the vesicle and its coronal layer formed by surface decorations, has become routine. For exosomes, the smallest of EVs, the relative difference between the hydrodynamic and vesicles sizes is significant. The characterization of vesicles sizes by the cryogenic transmission electron microscopy (cryo-TEM) imaging, a gold standard technique, remains a challenge due to the cost of the instrument, the expertise required to perform the sample preparation, imaging and data analysis, and a small number of particles often observed in images. A widely available and accessible alternative is the atomic force microscopy (AFM), which can produce versatile data on three-dimensional geometry, size, and other biophysical properties of extracellular vesicles. The developed protocol guides the users in utilizing this analytical tool and outlines the workflow for the analysis of EVs by the AFM, which includes the sample preparation for imaging EVs in hydrated or desiccated form, the electrostatic immobilization of vesicles on a substrate, data acquisition, its analysis, and interpretation. The representative results demonstrate that the fixation of EVs on the modified mica surface is predictable, customizable, and allows the user to obtain sizing results for a large number of vesicles. The vesicle sizing based on the AFM data was found to be consistent with the cryo-TEM imaging.&#160;

A step-by-step procedure is described for label-free immobilization of exosomes and extracellular vesicles from liquid samples and their imaging by atomic force microscopy (AFM). The AFM images are used to estimate the size of the vesicles in the solution and characterize other biophysical properties.&#160;

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane ...

Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the structure-function relationship of these receptors in situ, we need to visualize and track them on the live cell surface with enough spatiotemporal resolution. Here we show how to use recently developed Lattice Light-Sheet Microscopy (LLSM) to image T-cell receptors (TCRs) four-dimensionally (4D, space and time) at the live cell membrane. T cells are one of the main effector cells of the adaptive immune system, and here we used T cells as an example to show that the signaling and function of these cells are driven by the dynamics and interactions of the TCRs. LLSM allows for 4D imaging with unprecedented spatiotemporal resolution. This microscopy technique therefore can be generally applied to a wide array of surface or intracellular molecules of different cells in biology.

The goal of this protocol is to show how to use Lattice Light-Sheet Microscopy to four-dimensionally visualize surface receptor dynamics in live cells. Here T cell receptors on CD4+ primary T cells are shown.

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the ...

Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis process, which maintains enzymes far from their substrate. Characterization of these complex interactions is thus a challenge in complex substrates such as LB. The method here measures molecular interactions between fluorophore-tagged molecules and native autofluorescent lignin, to be revealed by F&#246;rster resonance energy transfer (FRET). Contrary to FRET measurements in living cells using two exogenous fluorophores, FRET measurements in plants using lignin is not trivial due to its complex autofluorescence. We have developed an original acquisition and analysis pipeline with correlated observation of two complementary properties of fluorescence: fluorescence emission and lifetime. sFLIM (spectral and fluorescent lifetime imaging microscopy) provides the quantification of these interactions with high sensitivity, revealing different interaction levels between biomolecules and lignin.

This protocol describes an original setup combining spectral and fluorescence lifetime measurements to evaluate F&#246;rster resonance energy transfer (FRET) between rhodamine-based fluorescent probes and lignin polymer directly in thick plant sections.

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis ...

Optical Tweezers to Study RNA-Protein Interactions in Translation Regulation

RNA adopts diverse structural folds, which are essential for its functions and thereby can impact diverse processes in the cell. In addition, the structure and function of an RNA can be modulated by various trans-acting factors, such as proteins, metabolites or other RNAs. Frameshifting RNA molecules, for instance, are regulatory RNAs located in coding regions, which direct translating ribosomes into an alternative open reading frame, and thereby act as gene switches. They may also adopt different folds after binding to proteins or other trans-factors. To dissect the role of RNA-binding proteins in translation and how they modulate RNA structure and stability, it is crucial to study the interplay and mechanical features of these RNA-protein complexes simultaneously.&#160;This work illustrates how to employ single-molecule-fluorescence-coupled optical tweezers to explore the conformational and thermodynamic landscape of RNA-protein complexes at a high resolution. As an example, the interaction of the SARS-CoV-2 programmed ribosomal frameshifting element with the trans-acting factor short isoform of zinc-finger antiviral protein is elaborated. In addition, fluorescence-labeled ribosomes were monitored using the confocal unit, which would ultimately enable the study of translation elongation. The fluorescence coupled OT assay can be widely applied to explore diverse RNA-protein complexes or trans-acting factors regulating translation and could facilitate studies of RNA-based gene regulation.

This protocol presents a complete experimental workflow for studying RNA-protein interactions using optical tweezers. Several possible experimental setups are outlined including the combination of optical tweezers with confocal microscopy.

RNA adopts diverse structural folds, which are essential for its functions and thereby can impact diverse processes in the cell. In addition, the ...

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, ...