Name: aptamer-based target detection facilitated by a 3-stage g-quadruplex isothermal exponential amplification reaction
Uploaded: 2022-10-06T13:40:00
Description: Watch this Scientific Journal Video about Aptamer-Based Target Detection Facilitated by a 3-Stage G-Quadruplex Isothermal Exponential Amplification Reaction at JoVE.com

This research was supported by the Research and Development Funding from Aptagen LLC.

See Supplementary Table 1 (the reaction set-up table) for tube preparation, including the specific volumes and concentrations of the reaction components. The protocol demonstrated here uses a preassembled detection platform kit as described in the Table of Materials. All the components must be kept on ice unless indicated otherwise.
1. Preparation of ribozyme
NOTE: The primary detection component is an allosteric (apt...

<ol>
	<li>Ellington, A. D., Szostak, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+selection+of+RNA+molecules+that+bind+specific+ligands.">In vitro selection of RNA molecules that bind specific ligands.</a> Nature. 346 (6287), 818-822 (1990).</li><li>Tang, J., Breaker, R. R. <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+allosteric+ribozymes.">Rational design of allosteric ribozymes.</a> Chemical Biology. 4 (6), 453-459 (1997).</li><li>Stoltenburg, R., Reinemann, C., Strehlitz, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SELEX--A+(r)evolutionary+method+to+generate+high-affinity+nucleic+acid+ligands.">SELEX--A (r)evolutionary method to generate high-affinity nucleic acid ligands.</a> Biomolecular Engineering. 24 (4), 381-403 (2007).</li><li>Liao, A. 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+simple+colorimetric+system+for+detecting+target+antigens+by+a+three-stage+signal+transformation-amplification+strategy.">A simple colorimetric system for detecting target antigens by a three-stage signal transformation-amplification strategy.</a> Biochemistry. 57 (34), 5117-5126 (2018).</li><li>Soukup, G. A., Emilsson, G. A., Breaker, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altering+molecular+recognition+of+RNA+aptamers+by+allosteric+selection.">Altering molecular recognition of RNA aptamers by allosteric selection.</a> Journal of Molecular Biology. 298 (4), 623-632 (2000).</li><li>Van Ness, J., Van Ness, L. K., Galas, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isothermal+reactions+for+the+amplification+of+oligonucleotides.">Isothermal reactions for the amplification of oligonucleotides.</a> Proceedings of the National Academy of Sciences of the United States of America. 100 (8), 4504-4509 (2003).</li><li>Cheng, X., Liu, X., Bing, T., Cao, Z., Shangguan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+peroxidase+activity+of+G-quadruplex-hemin+complexes+and+its+application+in+ligand+screening.">General peroxidase activity of G-quadruplex-hemin complexes and its application in ligand screening.</a> Biochemistry. 48 (33), 7817-7823 (2009).</li><li>Nie, 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=Reporter-triggered+isothermal+exponential+amplification+strategy+in+ultrasensitive+homogeneous+label-free+electrochemical+nucleic+acid+biosensing.">Reporter-triggered isothermal exponential amplification strategy in ultrasensitive homogeneous label-free electrochemical nucleic acid biosensing.</a> Chemical Communications. 50 (47), 6211-6213 (2014).</li><li>Tabuchi, T., Yokobayashi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+riboswitches.">Cell-free riboswitches.</a> RSC Chemical Biology. 2 (5), 1430-1440 (2021).</li><li>Ao, 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=Integration+of+an+expression+platform+in+the+SELEX+cycle+to+select+DNA+aptamer+binding+to+a+disease+biomarker.">Integration of an expression platform in the SELEX cycle to select DNA aptamer binding to a disease biomarker.</a> ACS Omega. 7 (12), 10804-10811 (2022).</li></ol>

The method presented here takes advantage of the transition between an initially disordered secondary structure in an allosteric ribozyme and the additional stability conferred through the binding of the target to the aptameric domain to activate the cis-cleaving hammerhead ribozyme. The stability of the ribozyme was adjusted to minimize catalytic activity in the absence of the target while allowing target binding to restore the active structure. Additionally, care must be taken to balance the buffers of the multiple enz...

Aptamers are typically single-stranded DNA or RNA selected through an evolutionary process with high binding affinity and specificity to the desired targets1. In addition to binding ability, aptamers can be linked to and control motifs with signal-output functions2,3, amplifying said signal and improving the system&#39;s sensitivity. The G-quadruplex isothermal exponential amplification reaction (GQ-EXPAR) system is a three-part system (Figure 1) that develops a visual signal as successive components are added to a single reaction vessel to produce a visual output4. This system allows for the detection of a specific target, herein theophylline, in a given sample within 15 min using a streamlined workflow to allow for fast, specific detection of a target of interest. This method must be considered for samples where specific quantification of the target concentration is a lower concern than high specificity of the response in a short amount of time.
An allosteric riboswitch, a structure-switching RNA molecule (ribozyme) that undergoes self-cleavage, produces the initial signal. This construct is based on the hammerhead ribozyme, with an aptameric domain introduced in Stem II as a regulator of cleavage activity. Its self-cleavage function is activated when its aptameric domain is stabilized on binding to its target5. Otherwise, the switch is inactive in its native state.
The subsequent exponential amplification reaction (EXPAR) uses the release of the self-cleaved RNA strand from the first stage to prime an isothermal amplification reaction6. The amplification product of EXPAR has peroxidase activity7, acting as the basis for the last stage of the system. When some substrates are oxidized in conjunction with peroxide breakdown, they produce a fluorescent output that can be measured on various instrumentation. Other common substrates can be substituted to produce a colored product for visual detection. EXPAR and the peroxidase activity of its amplification products act as a 2-stage signal-enhancer, increasing sensitivity to greater levels compared to conventional strategies7,8.
The detection of theophylline versus caffeine is used as an example of the specificity of this detection platform, as they differ by only a single methyl group (Figure 2). This demonstration of the system produces a colorimetric output for visual detection of at least 500 nM theophylline.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3,3&#39;,5,5&#39;-Tetramethylbenzidine dihydrochloride (TMB) solution with H2O2, &#34;MaxSignal TMB Microwell Substrate Solution&#34;</td>
			<td>PerkinElmer</td>
			<td>FOOD-1806-1000</td>
			<td>Color development reagent. Minimize exposure to light and atmosphere. Previously BIOO Scientific catalog number 1806. 
			Supplied in Apta-beacon Demonstration Kit as Tube 3-2.</td>
		</tr>
		<tr>
			<td>5&#8217;- TCC CTC CCT CCC TCC CAG TCC AGA CTC TTC CCT CCC TCC CTC CCA GA-Biotin-3&#8217;</td>
			<td>Integrated DNA Technologies (IDT)</td>
			<td>Custom</td>
			<td>Optimized QtQ47 DNA template for EXPAR to produce G-quadruplex, primed by G-quadruplex. This is used for the &#34;exponential&#34; part of EXPAR, to rapidly increase the amount of DNAzyme used for Step 3 color development. Template includes a 3&#39;-biotin to prevent unintended extension by Bst 2.0 DNA polymerase during EXPAR. 
			Part of Apta-beacon Demonstration Kit Tube 2-2.</td>
		</tr>
		<tr>
			<td>5&#8217;-GGG AAC UAU ACA ACC UAG GGC GAC CCU GAU GAG CCU UAU ACC AGC CGA AAG GCC CUU GGC AGA CGU UGA AAC GGU GAA AGC CGU AGG UUG CCC UAG GUU GUA UAG UU-3&#8217;</td>
			<td>Integrated DNA Technologies (IDT)</td>
			<td>Custom</td>
			<td>Theophylline-recognizing allosteric ribozyme sequence (self-cleaving ribozyme regulated by RNA aptamer domain for theophylline) modified from Soukup et al. 
			Soukup, G. A., Emilsson, G. A., and Breaker, R. R. (2000) Altering molecular recognition of RNA aptamers by allosteric selection, Journal of molecular biology 298, 623-632. 
			Supplied in Apta-beacon Demonstration Kit as Tube 1-3.</td>
		</tr>
		<tr>
			<td>5&#8217;-TCC CTC CCT CCC TCC CAG TCC AGA CTC TAC GGC TTT CAC CGT TTC AAC G-Biotin-3&#8217;</td>
			<td>Integrated DNA Technologies (IDT)</td>
			<td>Custom</td>
			<td>Optimized P3tQ49 DNA template for EXPAR to produce G-quadruplex, primed by P3 cleavage product. This is used in Step 2 to translate the cleavage product into DNAzyme used for Step 3 color development. Template includes a 3&#39;-biotin to prevent unintended extension by Bst 2.0 DNA polymerase during EXPAR. 
			Part of Apta-beacon Demonstration Kit Tube 2-2.</td>
		</tr>
		<tr>
			<td>5x Ribozyme Buffer</td>
			<td>Aptagen, LLC</td>
			<td>N/A</td>
			<td>5x composition: 600 mM Tris-HCl (pH 7.5), 150 mM MgCl2, 25 mM DTT. Used in Step 1. 
			Supplied in Apta-beacon Demonstration Kit as Tube 1-4.</td>
		</tr>
		<tr>
			<td>Apta-beaconTM (GQ-EXPAR, TMB) Demonstration Kit</td>
			<td>Aptagen, LLC</td>
			<td>GQ-EXPAR-TMB</td>
			<td>The demo kit showcases the specificity of the colorimetric assay by detecting difficult small molecule targets, theophylline versus caffeine, which only differ by a single methyl group.&#160;</td>
		</tr>
		<tr>
			<td>Bst 2.0 DNA polymerase</td>
			<td>New England Biolabs</td>
			<td>M0537S</td>
			<td>Isothermal amplification polymerase with strand-displacement activity. Part of the Nickase-Polymerase Mix, prepared at 9.375 U/uL. 
			Part of Apta-beacon Demonstration Kit Tube 2-1.</td>
		</tr>
		<tr>
			<td>Buffer 3.1</td>
			<td>New England Biolabs</td>
			<td>B6003SVIAL</td>
			<td>Combined with 2 uL of 10X Isothermal Amplification Buffer and 27.5 uL of nuclease-free water to produce 1.11X EXPAR Buffer in EXPAR Mix. This product replaces the previously-used B7203SVIAL that was part of the initial system development (same composition except non-recombinant BSA). 
			Part of Apta-beacon Demonstration Kit Tube 2-2.</td>
		</tr>
		<tr>
			<td>Caffeine</td>
			<td>Sigma-Aldrich</td>
			<td>C0750-100G</td>
			<td>Aptamer counter-target (control), prepared with nuclease-free water. 
			Supplied in Apta-beacon Demonstration Kit as Tube 1-1.</td>
		</tr>
		<tr>
			<td>dNTPs</td>
			<td>New England Biolabs</td>
			<td>N0447S</td>
			<td>Part of the EXPAR reaction mixture. 
			Part of Apta-beacon Demonstration Kit Tube 2-2.</td>
		</tr>
		<tr>
			<td>EXPAR reaction mixture</td>
			<td>Aptagen, LLC</td>
			<td>N/A</td>
			<td>0.44 mM dNTPs, 0.38 &#956;M P3tQ49, 0.38 &#956;M QtQ47, 44.44 mM Tris-HCl (pH 8.4), 63.5 mM NaCl, 31.5 mM KCl, 6.35 mM MgCl2, 1.27 mM MgSO4, 6.35 mM (NH4)2SO4, 63.5 &#956;g/ml BSA, 0.0635 % Tween 20. 
			Part of Apta-beacon Demonstration Kit Tube 2-2.</td>
		</tr>
		<tr>
			<td>Hemin</td>
			<td>Sigma-Aldrich</td>
			<td>H9039</td>
			<td>Resuspended in DMF to a final concentration of 25 uM. 
			Supplied in Apta-beacon Demonstration Kit as Tube 3-1.</td>
		</tr>
		<tr>
			<td>Isothermal Amplification Buffer</td>
			<td>New England Biolabs</td>
			<td>B0537SVIAL</td>
			<td>Combined with 2 uL of 10x Buffer 3.1 and 27.5 &#181;L of nuclease-free water to produce 1.11x EXPAR Buffer in EXPAR Mix. 
			Part of Apta-beacon Demonstration Kit Tube 2-2.</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Amresco (VWR)</td>
			<td>E525-500ML</td>
			<td>Hammerhead ribozyme cofactor, necessary for self-cleavage. 
			Part of Apta-beacon Demonstration Kit Tube 1-3.</td>
		</tr>
		<tr>
			<td>MJ PTC-100 Thermocycler</td>
			<td>MJ Research, Inc.</td>
			<td>PTC-100</td>
			<td>Thermocycler to control incubation temperatures. Any thermocycler or hot block can be used.</td>
		</tr>
		<tr>
			<td>N, N-Dimethylformamide (DMF)</td>
			<td>Sigma-Aldrich</td>
			<td>227056-100ML</td>
			<td>Used to resuspend hemin and maximize shelf life in freezer. 
			Part of Apta-beacon Demonstration Kit Tube 3-1.</td>
		</tr>
		<tr>
			<td>Nickase-polymerase Mix</td>
			<td>Aptagen, LLC</td>
			<td>N/A</td>
			<td>Nt.BstNBI (9.375 units/&#956;L) and Bst 2.0 DNA polymerase (0.5 units/&#956;L). 
			Supplied in Apta-beacon Demonstration Kit as Tube 2-1.</td>
		</tr>
		<tr>
			<td>Nt.BstNBI</td>
			<td>New England Biolabs</td>
			<td>R0607S</td>
			<td>Nicking enzyme to allow continued isothermal amplification. Part of the Nickase-Polymerase Mix, prepared at 0.5 U/uL. 
			Part of Apta-beacon Demonstration Kit Tube 2-1.</td>
		</tr>
		<tr>
			<td>T4 Kinase Buffer</td>
			<td>New England Biolabs</td>
			<td>B0201SVIAL</td>
			<td>Buffer for enzyme necessary to remove cyclic phosphate. 
			Part of Apta-beacon Demonstration Kit Tube 1-4.</td>
		</tr>
		<tr>
			<td>T4 polynucleotide kinase</td>
			<td>New England Biolabs</td>
			<td>M0201S</td>
			<td>Removes cyclic phosphate post-cleavage to allow cleavage product to prime isothermal amplification reaction. 
			Supplied in Apta-beacon Demonstration Kit as PCR Tubes.</td>
		</tr>
		<tr>
			<td>Tecan GENios FL</td>
			<td>Tecan</td>
			<td>Genios-FL TWT</td>
			<td>Plate reader to measure absorbance signal from Step 3 results.</td>
		</tr>
		<tr>
			<td>Theophylline</td>
			<td>Sigma-Aldrich</td>
			<td>T1633-50G</td>
			<td>Aptamer target, prepared with nuclease-free water. 
			Supplied in Apta-beacon Demonstration Kit as Tube 1-2.</td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>American Bioanalytical</td>
			<td>AB14043-01000</td>
			<td>Aptamer binding buffer. 
			Part of Apta-beacon Demonstration Kit Tube 1-4.</td>
		</tr>
	</tbody>
</table>

aptamer-based target detection facilitated by a 3-stage g-quadruplex isothermal exponential amplification reaction

Aptamers are target-recognition molecules that bind with high affinity and specificity. These characteristics can be leveraged to control other molecules with signal-generation capability. For the system described herein, target recognition through an aptameric domain, Stem II of a modified hammerhead ribozyme, activates the self-cleaving ribozyme by stabilizing the initially unstructured construct. The cis-cleaving RNA acts at the junction of Stem III and Stem I, creating two cleavage products. The longer cleavage product primes an isothermal exponential amplification reaction (EXPAR) of the two similar catalytically active G-quadruplexes. Those resulting amplification products catalyze peroxidase reduction, which is coupled to the reduction of a colorimetric substrate with an output that the naked eye can detect. The 3-part system described in the present study improves detection modalities such as enzyme-linked immunosorbent assays (ELISAs) by producing a visually detectable signal for indicating the presence of as low as 0.5 &#181;M theophylline in as little as 15 min.

The detection platform depicted in Figure 1 converts aptameric target recognition into visually distinct differences between the sample preparations (target vs. non-target, Figure 2) in a short period of time. An allosteric ribozyme identified by Soukup et al.5 served as the starting point for creating a less noisy sequence with response to the target over the control and negative samples. The optimized construct was able to recognize as ...

Watch this Scientific Journal Video about Aptamer-Based Target Detection Facilitated by a 3-Stage G-Quadruplex Isothermal Exponential Amplification Reaction at JoVE.com

Aptamer-Based Target Detection Facilitated by a 3-Stage G-Quadruplex Isothermal Exponential Amplification Reaction

The present protocol demonstrates the use of a fast, 3-stage, aptamer-based exponential amplification assay to detect targets. Sample preparation, signal amplification, and color development are covered to implement this system to recognize the presence of theophylline over that of caffeine.

Aptamers are target-recognition molecules that bind with high affinity and specificity. These characteristics can be leveraged to control other molecules ...

This method offers a way to amplify the signal from binding events into a form that is detectable visually for low resource use or can be quantified using absorbance-based instrumentation. This is a relatively quick, one-pot protocol to determine the presence of the aptamer's target in a sample of interest. Results can be compared against a standard calibration curve if quantification is desired.Individuals must be very precise when transferring and mixing the solutions, as insufficient mixing will lead to negative results. Also, ensure reagents and solutions are kept chilled until use to minimize background signal. Begin by activating the ribozyme cleavage products by adding five units of T4 polynucleotide kinase in a PCR tube.Then, add one microliter of pre-prepared 5X ribozyme buffer to each sample tube. To visually demonstrate specificity, add 1.5 microliters of two millimolar theophylline or two millimolar caffeine to each sample tube as the test samples and mix each sample thoroughly by pipetting five to 10 times. Next, add two microliters of 600 nanomolar RNA containing a target-specific aptameric domain to each sample tube, followed by pipetting to mix the components quickly.Now, place the tube on a cold block to minimize the background signal. After preparing all the reaction tubes, incubate each sample at room temperature for precisely three minutes. At the end of the incubation, return the sample tubes to ice immediately.Add 3.5 microliters of nicase polymerase enzyme mixture to each sample tube and mix well. Then, add 31.5 microliters of EXPAR reaction mixture containing template nucleotides and reaction buffer to each sample tube and mix by pipetting. Once all the samples are prepared, incubate the prepared sample at 55 degrees Celsius for five minutes using a timer.Immediately return the sample tubes to the ice after incubation. Add two microliters of hemin solution to each sample tube for color development and mix well. Then, add 58 microliters of the commercially available TMB solution to each sample tube, mix well, and incubate for color development.Read the samples qualitatively by eye or quantify the results using an absorbance plate reader, as described in the manuscript. The optimized construct could recognize as little as 500 nanomolar theophylline in 30 minutes. Sample preparations exposed to the target produce a blue color, while samples containing no target remain colorless.A standard curve was prepared from the signal measured at A450 after 30 minutes of color development. It is important to keep samples on ice when not carrying out incubations, as even the optimized RNA will still exhibit a low background activity level.

aptamer-based-target-detection-facilitated-3-stage-g-quadruplex

Research

JoVE Journal

Bioengineering

Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed secondary tumors1,2,3. CTCs are indicators of metastatic disease and their detection in blood samples may be used to diagnose cancer and monitor a patient&prime;s response to therapy. Since CTCs are rare, comprising about one tumor cell among billions of normal blood cells in advanced cancer patients, their detection and enumeration is a difficult task. We exploit the presence of pigment in most melanoma cells to generate photoacoustic, or laser induced ultrasonic waves in a custom flow cytometer for detection of circulating melanoma cells (CMCs)4,5. This process entails separating a whole blood sample using centrifugation and obtaining the white blood cell layer. If present in whole blood, CMCs will separate with the white blood cells due to similar density. These cells are resuspended in phosphate buffered saline (PBS) and introduced into the flowmeter. Rather than a continuous flow of the blood cell suspension, we induced two phase flow in order to capture these cells for further study. In two phase flow, two immiscible liquids in a microfluidic system meet at a junction and form alternating slugs of liquid6,7. PBS suspended white blood cells and air form microliter slugs that are sequentially irradiated with laser light. The addition of a surfactant to the liquid phase allows uniform slug formation and the user can create different sized slugs by altering the flow rates of the two phases. Slugs of air and slugs of PBS with white blood cells contain no light absorbers and hence, do not produce photoacoustic waves. However, slugs of white blood cells that contain even single CMCs absorb laser light and produce high frequency acoustic waves. These slugs that generate photoacoustic waves are sequestered and collected for cytochemical staining for verification of CMCs.

We have developed a flow cytometer using laser induced ultrasound to detect circulating melanoma cells as an early indicator of metastatic disease.

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed ...

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a model microorganism. The biosensor relies on direct binding of the target bacteria cells onto its surface, while no pretreatment (e.g.&#160;by cell lysis) of the studied sample is required. A mesoporous Si thin film is used as the optical transducer element of the biosensor. Under white light illumination, the porous layer displays well-resolved Fabry-P&#233;rot fringe patterns in its reflectivity spectrum. Applying a fast Fourier transform (FFT) to reflectivity data results in a single peak. Changes in the intensity of the FFT peak are monitored. Thus, target bacteria capture onto the biosensor surface, through antibody-antigen interactions, induces measurable changes in the intensity of the FFT peaks, allowing for a &#39;real time&#39; observation of bacteria attachment.
The mesoporous Si film, fabricated by an electrochemical anodization process, is conjugated with monoclonal antibodies, specific to the target bacteria. The immobilization, immunoactivity and specificity of the antibodies are confirmed by fluorescent labeling experiments. Once the biosensor is exposed to the target bacteria, the cells are directly captured onto the antibody-modified porous Si surface. These specific capturing events result in intensity changes in the thin-film optical interference spectrum of the biosensor. We demonstrate that these biosensors can detect relatively low bacteria concentrations (detection limit of 104 cells/ml) in less than an hour.

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor for rapid bacteria detection is introduced. The biosensor is based on a nanostructured porous Si, which is designed to directly capture the target bacteria cells onto its surface. We use monoclonal antibodies, immobilized onto the porous transducer, as the capture probes. Our studies demonstrate the applicability of these biosensors for the detection of low bacterial concentrations within minutes with no prior sample processing (such as cell lysis).

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a ...

Oscillation and Reaction Board Techniques for Estimating Inertial Properties of a Below-knee Prosthesis

The purpose of this study was two-fold: 1) demonstrate a technique that can be used to directly estimate the inertial properties of a below-knee prosthesis, and 2) contrast the effects of the proposed technique and that of using intact limb inertial properties on joint kinetic estimates during walking in unilateral, transtibial amputees. An oscillation and reaction board system was validated and shown to be reliable when measuring inertial properties of known geometrical solids. When direct measurements of inertial properties of the prosthesis were used in inverse dynamics modeling of the lower extremity compared with inertial estimates based on an intact shank and foot, joint kinetics at the hip and knee were significantly lower during the swing phase of walking. Differences in joint kinetics during stance, however, were smaller than those observed during swing. Therefore, researchers focusing on the swing phase of walking should consider the impact of prosthesis inertia property estimates on study outcomes. For stance, either one of the two inertial models investigated in our study would likely lead to similar outcomes with an inverse dynamics assessment.

Body segmental inertial properties are required for inverse dynamics modeling. Using an oscillation and reaction board technique, inertial properties of below-knee prostheses were measured. Using direct measures of prosthesis inertia in the inverse dynamics model of the prosthetic leg resulted in lower magnitudes of resultant joint forces and moments.

The purpose of this study was two-fold: 1) demonstrate a technique that can be used to directly estimate the inertial properties of a below-knee ...

Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement (EFIRM)

Exosomes are microvesicular structures that play a mediating role in intercellular communication. It is of interest to study the internal cargo of exosomes to determine if they carry disease discriminatory biomarkers. For performing exosomal analysis, it is necessary to develop a method for extracting and analyzing exosomes from target biofluids without damaging the internal content.
Electric field-induced release and measurement (EFIRM) is a method for specifically extracting exosomes from biofluids, unloading their cargo, and testing their internal RNA/protein content. Using an anti-human CD63 specific antibody magnetic microparticle, exosomes are first precipitated from biofluids. Following extraction, low-voltage electric cyclic square waves (CSW) are applied to disrupt the vesicular membrane and cause cargo unloading. The content of the exosome is hybridized to DNA primers or antibodies immobilized on an electrode surface for quantification of molecular content.
The EFIRM method is advantageous for extraction of exosomes and unloading cargo for analysis without lysis buffer. This method is capable of performing specific detection of both RNA and protein biomarker targets in the exosome. EFIRM extracts exosomes specifically based on their surface markers as opposed to size-based techniques.
Transmission electron microscopy (TEM) and assay demonstrate the functionality of the method for exosome capture and analysis. The EFIRM method was applied to exosomal analysis of 9 mice injected with human lung cancer H640 cells (a cell line transfected to express the exosome marker human CD63-GFP) in order to test their exosome profile against 11 mice receiving saline controls. Elevated levels of exosomal biomarkers (reference gene GAPDH and protein surface marker human CD63-GFP) were found for the H640 injected mice in both serum and saliva samples. Furthermore, saliva and serum samples were demonstrated to have linearity (R = 0.79). These results are suggestive for the viability of salivary exosome biomarkers for detection of distal diseases.

Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement EFIRM

Exosomes are microvesicular structures found within biofluids that potentially carry important disease discriminatory biomarkers. Here, a novel method is used to specifically extract exosomes and rapidly test the exosomal cargo for both RNA/protein targets following the disruption of exosomes using non-uniform electric cyclic square waves.

Exosomes are microvesicular structures that play a mediating role in intercellular communication. It is of interest to study the internal cargo of ...

Large-area Scanning Probe Nanolithography Facilitated by Automated Alignment and Its Application to Substrate Fabrication for Cell Culture Studies

Scanning probe microscopy has enabled the creation of a variety of methods for the constructive (&#39;additive&#39;) top-down fabrication of nanometer-scale features. Historically, a major drawback of scanning probe lithography has been the intrinsically low throughput of single probe systems. This has been tackled by the use of arrays of multiple probes to enable increased nanolithography throughput. In order to implement such parallelized nanolithography, the accurate alignment of probe arrays with the substrate surface is vital, so that all probes make contact with the surface simultaneously when lithographic patterning begins. This protocol describes the utilization of polymer pen lithography to produce nanometer-scale features over centimeter-sized areas, facilitated by the use of an algorithm for the rapid, accurate, and automated alignment of probe arrays. Here, nanolithography of thiols on gold substrates demonstrates the generation of features with high uniformity. These patterns are then functionalized with fibronectin for use in the context of surface-directed cell morphology studies.

Here we present a protocol for wide-area scanning probe nanolithography enabled by the iterative alignment of probe arrays, as well as the utilization of lithographic patterns for cell-surface interaction studies.

Scanning probe microscopy has enabled the creation of a variety of methods for the constructive (&#39;additive&#39;) top-down fabrication of ...

A Droplet-Based Microfluidic Approach and Microsphere-PCR Amplification for Single-Stranded DNA Amplicons

Droplet-based microfluidics enable the reliable production of homogeneous microspheres in the microfluidic channel, providing controlled size and morphology of the obtained microsphere. A microsphere copolymerized with an acrydite-DNA probe was successfully fabricated. Different methods such as asymmetric PCR, exonuclease digestion, and isolation on streptavidin-coated magnetic beads can be used to synthesize single-stranded DNA (ssDNA). However, these methods cannot efficiently use large amounts of highly purified ssDNA. Here, we describe a microsphere-PCR protocol detailing how ssDNA can be efficiently amplified and separated from dsDNA simply by pipetting from a PCR reaction tube. The amplification of ssDNA can be applied as potential reagents for the DNA microarray and DNA-SELEX (Systematic evolution of ligands by exponential enrichment) processes.

This work provides a method for the fabrication of droplet-based microfluidic platforms and the application of polyacrylamide microspheres for microsphere-PCR amplification. The microsphere-PCR method makes it possible to obtain single-stranded DNA amplicons without separating double-stranded DNA.

Droplet-based microfluidics enable the reliable production of homogeneous microspheres in the microfluidic channel, providing controlled size and ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Metabolic Profiling to Determine Bactericidal or Bacteriostatic Effects of New Natural Products using Isothermal Microcalorimetry

Due to the global threat of rising antimicrobial resistance, novel antibiotics are urgently needed. We investigate natural products from Myxobacteria as an&#160;innovative source of such new compounds. One bottleneck in the process is typically the elucidation of their mode-of-action. We recently established isothermal microcalorimetry as part of a routine profiling pipeline. This technology allows for investigating the effect of antibiotic exposure on the total bacterial metabolic response, including processes that are decoupled from biomass formation. Importantly, bacteriostatic and bactericidal effects are easily distinguishable without any user intervention during the measurements. However, isothermal microcalorimetry is a rather new approach and applying this method to different bacterial species usually requires pre-evaluation of suitable measurement conditions. There are some reference thermograms available of certain bacteria, greatly facilitating interpretation of results. As the pool of reference data is steadily growing, we expect the methodology to have increasing impact in the future and expect it to allow for in-depth fingerprint analyses enabling the differentiation of antibiotic classes.

The elucidation of the mode-of-action of a novel antibiotic is a challenging task in the drug discovery process. The goal of the method described here is the application of isothermal microcalorimetry using calScreener in antibacterial profiling to provide additional insight into drug-microbe interactions.

Due to the global threat of rising antimicrobial resistance, novel antibiotics are urgently needed. We investigate natural products from Myxobacteria as ...

Open-Source Miniature Fluorimeter to Monitor Real-Time Isothermal Nucleic Acid Amplification Reactions in Resource-Limited Settings

Traditional methods to detect and quantify nucleic acids rely on polymerase chain reaction (PCR) and require the use of expensive thermocyclers with integrated fluorescence detection of amplicons. Isothermal nucleic acid amplification technologies eliminate the need for thermal cycling; however, fluorescence-based detection of products is still required for real-time, quantitative results. Several portable isothermal heaters with integrated fluorescence detection are now commercially available; however, the cost of these devices remains a significant barrier to widespread adoption in resource-limited settings. Described here is a protocol for the design and assembly of a modular, low-cost fluorimeter constructed from off-the-shelf components. Enclosed in a compact 3D printed housing, the fluorimeter is designed to be placed atop a commercially available heat block holding a PCR tube. The fluorimeter described here was optimized to detect fluorescein isothiocyanate (FITC) dye, but the system can be modified for use with dyes commonly used as reporters in real-time nucleic acid amplification reactions. Clinical applicability of the system is demonstrated by performing real-time nucleic acid detection with two isothermal amplification technologies: recombinase polymerase amplification (RPA) for detection of positive control DNA provided in a commercial kit and reverse transcription loop-mediated isothermal amplification (RT-LAMP) for detection of clinically meaningful levels of SARS-CoV-2 RNA.

Detailed instructions are provided to build an open-source, modular fluorimeter that is compatible with many low-cost heaters to perform real-time, quantitative isothermal nucleic acid amplification.

Traditional methods to detect and quantify nucleic acids rely on polymerase chain reaction (PCR) and require the use of expensive thermocyclers with ...