Maria Baumgarten (IQ Biotechnology Platform, Infection Medicine, Lund University) is acknowledged for performing and providing scanning electron micrographs. This work was supported by grants from The Swedish Research Council Formas (2017-00100) as part of the European Third Joint Programming Initiative on Antimicrobial Resistance (JPIAMR) call &#34;Transmission Dynamics.&#34;&#160;The funders had no role in the study design, interpretation, writing, preparation of the manuscript, decision to submit, or decision to publish the work.

1. Modification of Glass Cover Slips (Template Stamps)
<ol>
	<li>To clean the glass cover slips, immerse them sequentially in 10 mL of 1.0 M HCl, deionized water, and 1.0 M of NaOH, respectively, for 10 min in each step in an ultrasonic cleaner at room temperature.
	<ol>
		<li>Dry the glass cover slips with nitrogen gas. 
		&#8203;NOTE: The cover slips are dried by evaporation under a stream of gaseous nitrogen.</li>
	</ol>
	</li>
	<li>Immerse the cleaned and dried cover slips in 10% (v/v), 10 mL solution of 3-amino-propyl-triethoxysilane (APTES) in ethanol for 1 h to introduce amino groups on the cover glass surface, at room temperature.
	<ol>
		<li>Rinse the cover slips with deionized water.</li>
		<li>Dry the cover slips with nitrogen gas.</li>
	</ol>
	</li>
	<li>Immerse the APTES-modified cover slips in 5% (v/v), 10 mL solution of glutaraldehyde in 10 mM phosphate buffer (pH 7.4) for 2 h in order to activate the amino groups on the surface, at room temperature6,15.
	<ol>
		<li>Rinse the cover slips with 10 mM phosphate buffer (pH 7.4) in order to remove excess glutaraldehyde from the surface.</li>
		<li>Dry the cover slips with nitrogen gas.</li>
	</ol>
	</li>
	<li>Prepare 1.0 mL of template (protein/bacteriophage) solution in 10 mM phosphate buffer (pH 7.4) in 0.1 mg/mL concentration. 
	&#8203;NOTE: Dissolve 0.1 mg of protein in 1.0 mL of phosphate buffer (10 mM, pH 7.4). If needed, a spectrophotometer (280 nm) may be used to determine the protein concentration.
	<ol>
		<li>Drop 200 &#181;L of this template solution onto the modified cover slips and incubate at 4 &#176;C overnight. 
		NOTE: The excess template solution can be used to prepare 1 - 2 more template stamps to be used in the characterization studies.</li>
		<li>Rinse the cover slips with 10 mM phosphate buffer (pH 7.4) in order to remove the unbound template from the surface. 
		NOTE: To rinse the electrodes, wash them with phosphate buffer (10 mM, pH 7.4) for 30 s.</li>
		<li>Dry the cover slips with nitrogen gas. 
		NOTE: The cover slips should be stored at 4 &#176;C until they are used in the polymerization step.</li>
	</ol>
	</li>
</ol>
2. Modification of Capacitive Gold Electrodes
<ol>
	<li>To clean the electrodes, immerse the electrodes in a small beaker, sequentially, containing 5 mL of ethanol (70%), deionized water, acetone, deionized water, acidic piranha solution (3:1, H2SO4:H2O2, v/v), and deionized water, respectively for 10 min in each step, in an ultrasonic cleaner at room temperature.
	<ol>
		<li>Dry the electrodes with nitrogen gas.</li>
	</ol>
	</li>
	<li>In order to perform the electropolymerization of tyramine, prepare 8 mL of 10 mM tyramine solution in 10 mM phosphate buffer (pH&#160;7.4) containing ethanol (2 mL)15. 
	&#8203;NOTE: The total volume of the tyramine solution must be 8 mL in total including 2 mL of ethanol.
	<ol>
		<li>Perform cyclic voltammetric scans (15 cycles) in this solution using a potentiostat covering a potential range of 0 - 1.5 V (Ag/AgCl) and a scan rate of 50 mV/s30.</li>
		<li>Rinse the electrodes with deionized water.</li>
		<li>Dry the electrodes with nitrogen gas.</li>
	</ol>
	</li>
	<li>Immerse the electrodes in a solution containing 30 mM acryloyl chloride and 30 mM trimethylamine in toluene (Vtotal = 5 mL) at room temperature overnight6,15,30.
	<ol>
		<li>Rinse the electrodes with deionized water. 
		NOTE: To ensure a better removal of unreacted acryloyl chloride and trimethylamine residues, it is also possible to wash the surface with NaOH after deionized water.</li>
		<li>Dry the electrodes with nitrogen gas.</li>
	</ol>
	</li>
</ol>
3. Preparation of Template Imprinted Capacitive Gold Electrodes
<ol>
	<li>Preparation of bacteriophage imprinted capacitive gold electrodes
	<ol>
		<li>Prior to polymerization, prepare a monomer solution containing monomer (N-hydroxymethyl acrylamide) and cross-linker (polyethylene glycol-400-dimethacrylate) in a ratio of 1:5 (mol/mol) in 1 mL of 10 mM phosphate buffer (pH 7.4). 
		NOTE: The monomer solution containing monomer and cross-linker can be used in different ratios, or the types of the monomer and cross-linker can be changed according to the template for optimization of the specific interaction.</li>
		<li>Add 1 mg of photo-initiator into this solution. 
		NOTE: If the polymerization is performed under UV light, then the photo-initiator must be used to initiate the polymerization. If the polymerization is performed by free radical polymerization, the type of the initiator must be changed.</li>
		<li>Pipette 1.5 &#181;L of this solution onto the gold surface of the modified gold electrode. 
		NOTE: The gold surface of the electrode is shown schematically in Figure 1.</li>
		<li>Bring the template stamp in contact with the monomer solution on the top of the gold electrode surface.</li>
		<li>Initiate the UV polymerization (365 nm, 400 W) and continue for 15 min31. 
		NOTE: UV polymerization is performed inside a cooling cabinet which is set to -25 &#176;C before initiating polymerization. Then, the UV light curing system is turned on and the polymerization is continued for 15 min before turning off the UV light curing system.</li>
		<li>Remove the template stamp from the surface by using forceps. 
		NOTE: While removing the template stamp from the surface, the polymeric film on the surface may be damaged. Therefore, the stamp should be removed from the surface very carefully and slowly without using force.</li>
		<li>Rinse the electrode surface with deionized water and dry it with nitrogen gas.</li>
		<li>Immerse the electrodes in 1 mL of 10 mM 1-dodecanethiol prepared in ethanol for 20 min in order to cover pinholes on the electrode surface.</li>
		<li>Rinse the electrodes with deionized water and dry the electrodes with nitrogen gas.</li>
	</ol>
	</li>
	<li>Preparation of Protein Imprinted Capacitive Gold Electrodes
	<ol>
		<li>Prior to polymerization, prepare a monomer solution containing monomers (acrylamide: 54 mg; N-hydroxymethylacrylamide: 140 &#181;L; N-isopropylacrylamide: 85.6 mg) and cross-linker (methylenebisacrylamide: 9.5 mg) in 820 &#181;L of ultra-pure water30,32.</li>
		<li>Prepare 5% (v/v) N,N,N&#39;,N&#39;-tetramethylethyldiamine (TEMED) in ultra-pure water.</li>
		<li>Add 20 &#181;L of the TEMED solution into the monomer solution and purge with nitrogen gas for 5 min.</li>
		<li>Prepare 10% (w/v) ammonium persulphate (APS) in ultra-pure water.</li>
		<li>Add 20 &#181;L of the APS solution into the monomer solution.</li>
		<li>Pipette 1.5 &#181;L of the monomer solution onto the modified gold electrode surface.</li>
		<li>Bring the template stamp into contact with the monomer-treated surface.</li>
		<li>Start polymerization at room temperature and continue for 5 h. 
		NOTE: For the preparation of protein-imprinted gold electrodes, instead of UV-polymerization, free-radical polymerization at room temperature (25 &#176;C) is performed by using APS-TEMED as the initiator-catalyst.</li>
		<li>Remove the template stamp from the surface carefully by using forceps.</li>
		<li>Rinse the electrode with deionized water and dry with nitrogen gas.</li>
		<li>Immerse the electrodes in 1 mL of 10 mM 1-dodecanethiol prepared in ethanol for 20 min in order to cover pinholes on the electrode surface.</li>
		<li>Rinse the electrodes with deionized water and dry the electrodes with nitrogen gas.</li>
	</ol>
	</li>
</ol>
4. Characterization of the Electrode Surface with Scanning Electron Microscopy (SEM)
<ol>
	<li>Mount the specimens on aluminum holders with adhesive carbon tape.</li>
	<li>Coat the electrodes with 10 nm palladium/gold.</li>
	<li>Examine the electrodes with SEM.</li>
</ol>
5. Real-time Capacitive Measurements with Template Imprinted Capacitive Gold Electrodes
<ol>
	<li>Insert the imprinted capacitive gold electrodes into the electrochemical flow cell integrated to a capacitive biosensor.</li>
	<li>Prepare 100 mL of regeneration buffer (25 mM glycine-HCl, pH 2.5, including 50 mM Tween-20) and 1 L running buffer (for bacteriophage imprinted system: 10 mM phosphate, pH 7.4; for protein-imprinted system: 50 mM Tris-HCl, pH 7.4).</li>
	<li>Start the analysis with the injection of regeneration buffer to regenerate the system and running buffer to re-equilibrate the system for 25 min.</li>
	<li>Prepare standard template solutions in the desired concentration range in running buffer. 
	NOTE: First prepare a stock of the template solution by dissolving 0.1 mg protein, or approximately 108 pfu bacteriophages, in 1.0 mL phosphate buffer (10 mM phosphate, pH 7.4). Then, prepare the standard solutions for the calibration curve by making ten sequential 10-fold dilutions from the stock solution. These solutions will be analyzed with the capacitive biosensor in step 5.5. Protein concentration can be measured by using a spectrophotometer (280 nm). In order to measure the bacteriophage concentration, a double layer agar method can be used, which is described in details previously19.</li>
	<li>Inject 250 &#181;L of these standard solutions sequentially to the system in optimum conditions (flow rate: 100 &#181;L/min, temperature: 25 &#176;C). 
	NOTE: In this application, the standard protein solutions were prepared in the concentration range of 1.0 x 10-4&#160;- 1.0 x 10-14, while the bacteriophage concentrations were in the range 1.0 x 101&#160;- 1.0 x 105 pfu/mL. The solutions were placed in the injection valve, and sequentially injected into the system, injecting the samples in triplicates through the single syringe pump and the multiport valve. The decrease in capacitance after injecting the standard solutions arising from the binding of template to the imprinted cavities is monitored automatically by the instrument&#39;s software.</li>
</ol>

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A., 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+detection+of+small+quantities+of+specific+bacteria+using+phage-based+wireless+biosensors.">Rapid detection of small quantities of specific bacteria using phage-based wireless biosensors.</a> 2016 10th International Conference on Sensing Technology (ICST). , 1-5 (2016).</li><li>Park, M. -. K., Chin, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+Approach+of+a+Phage-Based+Magnetoelastic+Biosensor+for+the+Detection+of+Salmonella+enterica+serovar+Typhimurium+in+Soil.">Novel Approach of a Phage-Based Magnetoelastic Biosensor for the Detection of Salmonella enterica serovar Typhimurium in Soil.</a> Journal of Microbiology and Biotechnology. 26 (12), 2051-2059 (2016).</li><li>Mack, J. D., Yehualaeshet, T., Park, M. -. K., Tameru, B., Samuel, T., Chin, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phage-Based+Biosensor+and+Optimization+of+Surface+Blocking+Agents+to+Detect+Salmonella+typhimurium+on+Romaine+Lettuce.">Phage-Based Biosensor and Optimization of Surface Blocking Agents to Detect Salmonella typhimurium on Romaine Lettuce.</a> Journal of food safety. 37 (2), 12299 (2017).</li><li>Colomer-Lluch, M., Jofre, J., Muniesa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic+resistance+genes+in+the+bacteriophage+DNA+fraction+of+environmental+samples.">Antibiotic resistance genes in the bacteriophage DNA fraction of environmental samples.</a> Plos One. 6 (3), 17549 (2011).</li><li>Lood, R., Ert&#252;rk, G., Mattiasson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revisiting+antibiotic+resistance+spreading+in+wastewater+treatment+plants+-+bacteriophages+as+a+much+neglected+potential+transmission+vehicle.">Revisiting antibiotic resistance spreading in wastewater treatment plants - bacteriophages as a much neglected potential transmission vehicle.</a> Frontiers in microbiology. 8, (2017).</li></ol>

The authors have nothing to disclose.

When this method is carried out, there are some critical steps that must be considered while following the protocol. One critical step is the cleaning step with acidic piranha solution. Step 2.1 must not be more than 10 min. The template solution for step 1.4 must not exceed 0.1 mg/mL, since these values have been optimized previously. Cyclic voltametric scans must not exceed 15 cycles in order to obtain the optimum thickness. For step 3.1.3, 1.5 &#181;L is an optimized value. This value must not be higher for this specific type of electrode. If the UV curing system has a power of 400 W, then the polymerization must be performed for a maximum of 10 - 15 min. As soon as the APS (initiator) is added to the solution after TEMED, the subsequent step must be performed very quickly to avoid immediate polymerization (step 3.2.5).
One of the most critical steps is the removal of the template stamp from the surface after UV polymerization. If this step is not performed properly, there is a risk that the polymeric film on the surface of the electrode may be removed with the stamp. Therefore, it is recommended to immerse the electrode and the protein stamp on top in a solution of water after the polymerization, then remove the stamp very slowly and carefully from the surface (step 3.1.6).
Based on the template used, modifications in the type and ratio of monomers used (functional monomer and cross-linker) may be assessed in terms of generating higher sensitivity. This must be determined empirically. Further, the affinity binding of the template molecule usually involves several different interactions simultaneously. Therefore, this may lead to problems during the regeneration steps. If the bound template is not released from the surface properly, this may influence the reusability of the electrode for further analysis. These multipoint affinities may also result from binding via weaker interactions. In such systems, non-specific binding may take place, which can negatively influence the selectivity of the system16. These are common, and general, limitations of the method.
Apart from these specific limitations, there are many significant advantages of the discussed method over existing methods. While RIAs, ELISAs, and fluorometric measurements are very sensitive, they require the usage of labeled material (either template or detector), while the biosensor is completely label-free. These methods are also more expensive and time-consuming. A biosensor approach allows for rapid, parallel synthesis of MIPs in different compositions at the same time16. Since only a few microliters of monomer solution is required for preparation, the method is convenient when using expensive or otherwise limited monomers. Further, single MIP electrodes can be used for approximately 80 analyses without a significant decrease in the performance, which is significantly higher than other existing methods30. Existing methods also suffer, in varying degrees, of low sensitivity and selectivity, while the described method allows for detection and quantification of molecules in the pM range with high selectivity.
Due to the cost-effectiveness, the ease of operating the instrument, and the real-time sensitive detection in a short time compared to existing methods, the biosensors are very promising point-of care detection systems under field conditions; e.g., for environmental monitoring and for applications in developing countries. In many applications within diagnosis of disease, real-time, sensitive, selective, and rapid detection of a biomarker in a complex mixture such as serum is required15,25. Here, biosensors are superior to existing methods, in particular, due to their robustness and sensitivity. Specifically for the detection of infectious agents, bacteriophages are recently regarded as alternative biorecognition element for biosensors due to their host bacteria specificity33,34,35. Replacement of antibodies with bacteriophages is very promising in order to reduce the cost and increase the stability even further36. Such a system will also allow for the detection and quantification of specific phages in the environment and from clinical samples. Due to the prevalence of bacteriophages, and their ability to transduce bacteria with antibiotic resistance genes37,38, such a method may be valuable studying the spread of resistant bacterial.

The quantification of biomolecules is used in many different research fields within science, employing methods like the radioimmunoassay (RIA) or ELISA1. Some of these methods require a labeled reagent like a radioisotope or enzyme labeled antibody/antigen, which makes them labor-intensive and time-consuming with complex procedures2. Further, the robustness, selectivity, and sensitivity of these methods are not sufficient for all analyses; in particular they are not sufficient when attogram quantities need to be analyzed, rather than pictogram quantities3. For this purpose, biosensors have gained considerable interest4,5, in particular in combination with molecular imprinting for an increased robustness.
Molecular imprinting relies on creating cavities by polymerizing functional monomers around the template6, creating artificial recognition sites that perfectly resemble the template7. This technique has been used for several applications, including drug delivery systems and analytical separation, but also as biorecognition elements in biosensors8,9,10. However, there are still some difficulties in the design of molecularly imprinted polymers (MIPs) for macromolecular templates like proteins and cells11,12. Due to this, many researchers have focused on imprinting the template protein directly onto a substrate, thus creating a surface that will be recognized by the target protein12. This surface coating technique used for employing recognition cavities for large molecules and assemblies including proteins is called microcontact imprinting13,14. The general procedure of the method depends on the polymerization between two surfaces - a template stamp and a polymer support - upon which the template is adsorbed on one surface15,16, and brought into contact with the monomer-treated surface. By this way, a thin polymer film is formed on the support via UV-polymerization. Finally, the template is removed, leaving behind template specific cavities at the surface of the imprinted electrode. This method has some advantages including a reduced activity loss of the imprinted molecule, as well as requiring very small amounts of template molecules for the imprinting process16,17. Thus, these cost-effective, stable, sensitive, and selective surfaces can be created on the sensor surfaces, targeting any template of the user&#39;s choice.
The biosensor can be used for the detection of&#160;single proteins and&#160;much larger biomacromolecules, including viruses. A specific group of viruses gaining recent interest is the bacteriophage, which is a virus that infects bacteria. Quick and sensitive detection of bacteriophages is important during biotechnological and biopharmaceutical processes in order to determine the infections of bacterial cultures with bacteriophages18. The most commonly used biological assay for bacteriophage detection is the double layer agar method19, which is laborious and time consuming. Several attempts have been made to develop novel diagnostic tools for viruses (including bacteriophages) such as atomic force microscopy (AFM)20, interferometry21, electrochemistry22, and sensor system23,24. A lot of work has been focused on biosensors owing to their advantages as being easy to operate, highly sensitive, and capable of real-time measurement15,25. A specific type of biosensor is based on changes in capacitance. These capacitive biosensors are the electrochemical sensors that measure changes in the dielectric properties when an analyte interacts with a biorecognition element on the sensor surface, causing a decrease in the capacitance2,4. Capacitive biosensors have been used for the detection of various analytes like antigens, antibodies, proteins, and heavy metal ions6,26,27,28. These types of biosensors have many advantages like inherent rapidity, high sensitivity, simplicity, low cost, easy manipulation, and real-time measurement without labeling29.
The method described herein is aimed at enabling the detection and quantification of low-abundant biomolecules in highly complex samples, without the need of using any labeling. In particular, the technique is most useful in the atto-picogram range of biomolecules, where other commercially existing instruments fail to accurately quantitate their target.

<table><tbody><tr><td>Glass Cover slips</td><td>ThermoFisher</td><td>102222</td><td>protein stamp</td></tr><tr><td>HCl</td><td>Sigma-Aldrich</td><td>H1758-500ML</td><td>cleaning</td></tr><tr><td>NaOH</td><td>Sigma-Aldrich</td><td>72068-100ML</td><td>cleaning</td></tr><tr><td>Ultrasonic cleaner</td><td>Branson Ultrasonic</td><td>BRANSONIC M1800- E</td><td>cleaning</td></tr><tr><td>3-amino-propyl-triethoxysilane (APTES)</td><td>Sigma-Aldrich</td><td>A3648-100ML</td><td>modification</td></tr><tr><td>EtOH</td><td>Sigma-Aldrich</td><td>1009836010</td><td>rinsing/cleaning</td></tr><tr><td>glutaraldehyde</td><td>Sigma-Aldrich</td><td>G5882-100ML</td><td>cross-linker</td></tr><tr><td>acetone</td><td>Sigma-Aldrich</td><td>34850-1L-M</td><td>cleaning</td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>339741-100ML</td><td>piranha solution</td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>H1009-500ML</td><td>piranha solution</td></tr><tr><td>tyramine</td><td>Sigma-Aldrich</td><td>T90344-5G</td><td>modification</td></tr><tr><td>CompactStat</td><td>Ivium Technologies</td><td>CompactStat.h: 30mA@10V/3MHz</td><td>potentiostat</td></tr><tr><td>Platinum Counter Electrode Kit</td><td>Equilabrium</td><td>AFCTR5</td><td>potentiostat</td></tr><tr><td>Reference Electrode</td><td>Equilabrium</td><td>RREF0021</td><td>potentiostat</td></tr><tr><td>acryloyl chloride</td><td>EMD Millipore</td><td>8.00826.0100</td><td>modification</td></tr><tr><td>triethylamine</td><td>EMD Millipore</td><td>8.08352.0100</td><td>modification</td></tr><tr><td>toluene</td><td>Sigma-Aldrich</td><td>244511-100ML</td><td>modification</td></tr><tr><td>N-hydroxymethyl acrylamide</td><td>Sigma-Aldrich</td><td>245801-100G</td><td>functional monomer</td></tr><tr><td>poly ethylene glycol-400-dimethacrylate</td><td>Sigma-Aldrich</td><td>409510-250ML</td><td>cross-linker</td></tr><tr><td>2-Hydroxy-4&amp;prime;-(2-hydroxyethoxy)-2-methylpropiophenone</td><td>Sigma-Aldrich</td><td>410896-50G</td><td>functional monomer</td></tr><tr><td>UV polymerizator</td><td>Dymax</td><td>Dymax 5000ECE</td><td>UV-polymerization</td></tr><tr><td>forceps</td><td>Sigma-Aldrich</td><td>Z168777-1EA</td><td>consumable</td></tr><tr><td>1-dodecanethiol</td><td>Sigma-Aldrich</td><td>471364-100ML</td><td>blocking agent</td></tr><tr><td>acrylamide</td><td>Sigma-Aldrich</td><td>A3553-100G</td><td>functional monomer</td></tr><tr><td>N-hydroxymethylacrylamide</td><td>Sigma-Aldrich</td><td>245801-100G</td><td>functional monomer</td></tr><tr><td>N-isopropylacrylamide</td><td>Sigma-Aldrich</td><td>415324-50G</td><td>functional monomer</td></tr><tr><td>methylenebisacrylamide</td><td>Sigma-Aldrich</td><td>146072-500G</td><td>cross-linking monomer</td></tr><tr><td>N,N,N&#039;,N&#039;-tetrametyhlethyldiamine (TEMED)</td><td>Sigma-Aldrich</td><td>T9281-25ML</td><td>catalyst</td></tr><tr><td>ammonium persulphate</td><td>Sigma-Aldrich</td><td>A3678-25G</td><td>initiator</td></tr><tr><td>Capacitive biosensor</td><td>CapSenze</td><td></td><td>Equipment</td></tr><tr><td>Glycine</td><td>Merck</td><td>1042011000</td><td>regeneration buffer</td></tr><tr><td>Tween-20</td><td>Sigma-Aldrich</td><td>P9416-50ML</td><td>regeneration buffer</td></tr><tr><td>Trizma base</td><td>Sigma-Aldrich</td><td>93352-1KG</td><td>running buffer</td></tr><tr><td>Na2HPO4&amp;nbsp;&amp;bull; 2H2O</td><td>Calbiochem</td><td>567547-1KG</td><td>running buffer</td></tr><tr><td>NaH2PO4&amp;nbsp;&amp;bull; 2H2O</td><td>Calbiochem</td><td>567549-1KG</td><td>running buffer</td></tr><tr><td>DELPHI correlative light and electron microscope</td><td>Phenom-World</td><td></td><td>equipment</td></tr><tr><td>Capacitive gold electrodes</td><td>CapSenze Biosystems</td><td></td><td>consumables</td></tr><tr><td>2,2&#039;-azobis(2-methypropionitrile)</td><td>Sigma-Aldrich</td><td>441090-25G</td><td>photo-initiator</td></tr><tr><td>CapSenze Smart Software</td><td>CapSenze Biosystems</td><td></td><td>software program</td></tr></tbody></table>

ultrasensitive detection of biomarkers by using a molecular imprinting based capacitive biosensor

The ability to detect and quantitate biomolecules in complex solutions has always been highly sought-after within natural science; being used for the detection of biomarkers, contaminants, and other molecules of interest. A commonly used technique for this purpose is the Enzyme-linked Immunosorbent Assay (ELISA), where often one antibody is directed towards a specific target molecule, and a second labeled antibody is used for the detection of the primary antibody, allowing for the absolute quantification of the biomolecule under study. However, the usage of antibodies as recognition elements limits the robustness of the method; as does the need of using labeled molecules. To overcome these limitations, molecular imprinting has been implemented, creating artificial recognition sites complementary to the template molecule, and obsoleting the necessity of using antibodies for initial binding. Further, for even higher sensitivity, the secondary labeled antibody can be replaced by biosensors relying on the capacitance for the quantification of the target molecule. In this protocol, we describe a method to rapidly and label-free detect and quantitate low-abundant biomolecules (proteins and viruses) in complex samples, with a sensitivity that is significantly better than commonly used detection systems such as the ELISA. This is all mediated by molecular imprinting in combination with a capacitance biosensor.

By following the protocol, according to the schematic in Figure 1, a bare gold electrode will be imprinted with a template, representing the structure of a biomacromolecule. This electrode can be applied in a capacitive biosensor (Figure 2), allowing the stable application of a template onto the electrode, and the measurement of changes in capacitance upon binding of the template.
A schematic representation of the capacitive biosensor is shown in Figure 2. The centris pump, which is responsible for continuous injection of the running buffer (10 mM phosphate, pH 7.4), and the regeneration buffer (25 mM glycine-HCl, pH 2.5) during regeneration into the flow cell, can be seen clearly in the figure. The flow cell consists of working, reference, and counter electrodes. The injection valve is composed of standard protein/bacteriophage solutions, which are passing through the degasser first and then injected sequentially into the system. As soon as the solutions reach the working electrode inserted in the flow cell, the result is monitored in real-time. The capacitance values can be registered by following the sensorgrams on the computer screen.
Figure 3&#160;and Figure 4 depict the differences between the surface of bare and imprinted gold electrodes. This characterization step is important to ensure that there are polymeric cavities, seen as roughness on the surface of the electrode after imprinting. Apart from SEM, there are also other characterization methods including AFM, contact angle measurements, ellipsometry etc., that can be used to characterize the surface after imprinting. By this way, it can be ensured that the imprinting process is successful and that template cavities are formed on the surface. Within these cavities, the template can bind with high specificity and affinity.
After injecting the standard solutions into the capacitive system, an average of the last five readings was calculated automatically by the software, and the calibration graphs were obtained by plotting the change in capacitance vs. the concentration of the analyte. The decrease in registered capacitance arose from the binding of the template. The more molecules that bind to the gold electrode surface, the higher the reduction in total capacitance, according to the general principle of capacitive measurements. Figure 5 and Figure 6 show that with increasing concentration of the analyte, the &#916;C increases as expected. The dynamic range (between which is the concentration range where the system is useful for the detection of a specific target) and the limit of detection (LOD) can be evaluated by analyzing these graphs. According to Figure 6, the bacteriophage imprinted capacitive biosensor can detect bacteriophages in the concentration range of 101 - 105 pfu/mL, with an LOD value of 10&#160;pfu/mL in this study. Both Figure 5 and Figure 6 also highlight the necessity of measuring the calibration curve in the same interval required for the template concentration is supposed, since the linearity of the regression may vary over the concentrations (Figure 5), or have different slopes (Figure 6). It should also be noted that due to the low concentrations used, the system is quite sensitive to fluctuations (turbidity in sample, air draft, etc.), and therefore, it is recommended to run at least triplicates to reduce the potential of including outliers. For the same reason, the standard deviation can be quite significant for highly diluted samples, as seen in Figure 6.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57208/57208fig1.jpg" /> 
Figure 1. Schematic representation of the microcontact imprinting method. (A) Preparation of the glass cover slips (template stamps), (B) preparation of the capacitive gold electrodes, (C) microcontact imprinting of the template onto the gold electrode surface via UV-polymerization, and (D) removal of template from the electrode surface (Reproduced from Ert&#252;rk et al., Biotechnology Reports 2014 (3): 65 - 72 with permission). <a href="https://cloudflare.jove.com/files/ftp_upload/57208/57208fig1large.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/57208/57208fig2.jpg" /> 
Figure 2.&#160;Schematic representation of the capacitive biosensor. The general layout of the capacitive biosensor used in this study (Reproduced from Ert&#252;rk et al., Biotechnology Reports 2014 (3): 65 - 72 with permission). <a href="https://cloudflare.jove.com/files/ftp_upload/57208/57208fig2large.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/57208/57208fig3.jpg" /> 
Figure 3.&#160;Scanning Electron Microscopy of protein imprinted electrodes. SEM images of (A) a bare gold electrode (scale bar = 20 &#181;m), and (B) a protein imprinted capacitive gold electrode (scale bar = 10 &#181;m). <a href="https://cloudflare.jove.com/files/ftp_upload/57208/57208fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57208/57208fig4.jpg" /> 
Figure 4.&#160;Scanning Electron Microscopy of bacteriophage imprinted electrodes. SEM images of a bare gold electrode (scale bar = 20 &#181;m) (A), and a bacteriophage imprinted capacitive gold electrode in different magnifications (6600x, scale bar = 10 &#181;m) (B), and 11,500X, scale bar = 5 &#181;m) (C); arrows denote adhered bacteriophages). <a href="https://cloudflare.jove.com/files/ftp_upload/57208/57208fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57208/57208fig5.jpg" /> 
Figure 5.&#160;Effect of buffer composition for calibration graphs. Calibration graph showing the change in capacitance vs. a protein concentration in optimum conditions (Running Buffer: 50 mM Tris-HCl, pH 7.4; Regeneration Buffer: 25 mM glycine-HCl, pH 2.5 including 50 mM Tween-20; Flow rate: 100 &#181;L/min, Sample volume: 250 &#181;L; T: 25 &#176;C). <a href="https://cloudflare.jove.com/files/ftp_upload/57208/57208fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57208/57208fig6.jpg" /> 
Figure 6.&#160;Representative calibration curve for large biomacromolecules. Calibration graph that shows the change in capacitance vs. bacteriophage concentration in optimum conditions (Running Buffer: 10 mM phosphate, pH 7.4; Regeneration Buffer: 25 mM glycine-HCl, pH 2.5 including 50 mM Tween-20; Flow rate: 100 &#181;L/min, Sample volume: 250 &#181;L; T: 25 &#176;C) <a href="https://cloudflare.jove.com/files/ftp_upload/57208/57208fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Ultrasensitive Detection of Biomarkers by Using a Molecular Imprinting Based Capacitive Biosensor at JoVE.com

Ultrasensitive Detection of Biomarkers by Using a Molecular Imprinting Based Capacitive Biosensor

Here, we present a protocol for the detection and quantification of low abundant molecules in complex solutions using molecular imprinting in combination with a capacitance biosensor.

The ability to detect and quantitate biomolecules in complex solutions has always been highly sought-after within natural science; being used for the ...

ultrasensitive-detection-biomarkers-using-molecular-imprinting-based

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

11.9K Views.  Lund University. Here, we present a protocol for the detection and quantification of low abundant molecules in complex solutions using molecular imprinting in combination with a capacitance biosensor.

Video: Ultrasensitive Detection of Biomarkers by Using a Molecular Imprinting Based Capacitive Biosensor

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

Bioengineering

ELIME (Enzyme Linked Immuno Magnetic Electrochemical) Method for Mycotoxin Detection

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous mycotoxins in food commodities. In this protocol we show how to fabricate and interrogate an electrochemical competitive Enzyme linked immunomagnetic assay based on the use of magnetic beads as solid support for the immunochemical chain3 and screen printed electrodes as sensing platform.
Our method aims to determine the total amount of HT-2 and T-2 toxins, mycotoxins belonging to the trichothecenes family and of great concern for human health4. The use of an antibody clone with a cross reactivity of 100% towards HT-2 and T-2 allows to simultaneously detect both toxins with similar sensitivity5.
The first step of our assay is the coating step where we immobilize HT2-KLH conjugate toxin on the surface of magnetic beads. After a blocking step, necessary to avoid non-specific absorptions, the addition of a monoclonal antibody allows the competition between immobilized HT-2 and free HT-2 or T-2 present in the sample or dissolved in a standard solution.
At the end of the competition step, the amount of monoclonal antibody linked to the immobilized HT-2 will be inversely proportional to the amount of toxin in the sample solution.
A secondary antibody labeled with alkaline phosphatase (AP) is used to reveal the binding between the specific antibody and the immobilized HT-2. The final measurement step is performed by dropping an aliquot of magnetic bead suspension, corresponding to a specific sample/standard solution, on the surface of a screen-printed working electrode; magnetic beads are immobilized and concentrated by means of a magnet placed precisely under the screen-printed electrode. After two minutes of incubation between magnetic beads and a substrate for AP, the enzymatic product is detected by Differential Pulse Voltammetry (DPV) using a portable instrument (PalmSens) also able to initiate automatically eight measurements within an interval of few seconds.

ELIME Enzyme Linked Immuno Magnetic Electrochemical Method for Mycotoxin Detection

A protocol to detect trichothecenes (mycotoxins of concern for human health) using a newly developed screening method based on a competitive immunochemical method and a final electrochemical detection is demonstrated.

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous ...

Biology

Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform for reduced sample volume and assay time as well as full automation are required for potential use in point-of-care-diagnostics. Recently, we have demonstrated ultrasensitive detection of serum proteins, C-reactive protein (CRP) and cardiac troponin I (cTnI), utilizing a lab-on-a-disc composed of TiO2 nanofibrous (NF) mats. It showed a large dynamic range with femto molar (fM) detection sensitivity, from a small volume of whole blood in 30 min. The device consists of several components for blood separation, metering, mixing, and washing that are automated for improved sensitivity from low sample volumes. Here, in the video demonstration, we show the experimental protocols and know-how for the fabrication of NFs as well as the disc, their integration and the operation in the following order: processes for preparing TiO2 NF mat; transfer-printing of TiO2 NF mat onto the disc; surface modification for immune-reactions, disc assembly and operation; on-disc detection and representative results for immunoassay. Use of this device enables multiplexed analysis with minimal consumption of samples and reagents. Given the advantages, the device should find use in a wide variety of applications, and prove beneficial in facilitating the analysis of low abundant proteins.

This protocol demonstrates how to achieve femto molar detection sensitivity of proteins in 10 &#181;L of whole blood within 30 min. This can be achieved by using electrospun nanofibrous mats integrated in a lab-on-a-disc, which offers high surface area as well as effective mixing and washing for enhanced signal-to-noise ratio.

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform ...

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Exploring Biomolecular Interaction Between the Molecular Chaperone Hsp90 and Its Client Protein Kinase Cdc37 using Field-Effect Biosensing Technology

Biomolecular interactions play versatile roles in numerous cellular processes&#160;by regulating and coordinating functionally relevant biological events. Biomolecules such as proteins, carbohydrates, vitamins, fatty acids, nucleic acids, and enzymes are fundamental building blocks of living beings; they assemble into complex networks in biosystems to synchronize a myriad of life events. Proteins typically utilize complex interactome networks to carry out their functions; hence it is mandatory to evaluate such interactions to unravel their importance in cells at both cellular and organism levels. Toward this goal, we introduce a rapidly emerging technology, field-effect biosensing (FEB), to determine specific biomolecular interactions. FEB is a benchtop, label-free, and reliable biomolecular detection technique to determine specific interactions and uses high-quality electronic-based biosensors. The FEB technology can monitor interactions in the nanomolar range due to the biocompatible nanomaterials used on its biosensor surface. As a proof of concept, the protein-protein interaction (PPI) between heat shock protein 90 (Hsp90) and cell division cycle 37 (Cdc37) was elucidated. Hsp90 is an ATP-dependent molecular chaperone that plays an essential role in the folding, stability, maturation, and quality control of many proteins, thereby regulating multiple vital cellular functions. Cdc37 is regarded as a protein kinase-specific molecular chaperone, as it specifically recognizes and recruits protein kinases to Hsp90 to regulate their downstream signal transduction pathways. As such, Cdc37 is considered a co-chaperone of Hsp90. The chaperone-kinase pathway (Hsp90/Cdc37 complex) is hyper-activated in multiple malignancies promoting cellular growth; therefore, it is a potential target for cancer therapy. The present study demonstrates the efficiency of FEB technology using the Hsp90/Cdc37 model system. FEB detected a strong PPI between the two proteins (KD values of 0.014 &#181;M, 0.053 &#181;M, and 0.072 &#181;M in three independent experiments). In summary, FEB is a label-free and cost-effective PPI detection platform, which offers fast and accurate measurements.

Field-effect biosensing (FEB) is a label-free technique for detecting biomolecular interactions. It measures the electric current through the graphene biosensor to which the binding targets are immobilized. The FEB technology was used to evaluate biomolecular interactions between Hsp90 and Cdc37 and a strong interaction between the two proteins was detected.

Biomolecular interactions play versatile roles in numerous cellular processes&#160;by regulating and coordinating functionally relevant biological events. ...

Biochemistry

The Use of a &#946;-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions

Biosensors are becoming increasingly important and implemented in various fields such as pathogen detection, molecular diagnosis, environmental monitoring, and food safety control. In this context, we used &#946;-lactamases as efficient reporter enzymes in several protein-protein interaction studies. Furthermore, their ability to accept insertions of peptides or structured proteins/domains strongly encourages the use of these enzymes to generate chimeric proteins. In a recent study, we inserted a single-domain antibody fragment into the Bacillus licheniformis BlaP &#946;-lactamase. These small domains, also called nanobodies, are defined as the antigen-binding domains of single chain antibodies from camelids. Like common double chain antibodies, they show high affinities and specificities for their targets. The resulting chimeric protein exhibited a high affinity against its target while retaining the &#946;-lactamase activity. This suggests that the nanobody and &#946;-lactamase moieties remain functional. In the present work, we report a detailed protocol that combines our hybrid &#946;-lactamase system to the biosensor technology. The specific binding of the nanobody to its target can be detected thanks to a conductimetric measurement of the protons released by the catalytic activity of the enzyme.

In this work, we report a new method to study protein-protein interactions using a conductimetric biosensor based on the hybrid &#946;-lactamase technology. This method relies on release of protons upon hydrolysis of &#946;-lactams.

Biosensors are becoming increasingly important and implemented in various fields such as pathogen detection, molecular diagnosis, environmental ...

Using Extraordinary Optical Transmission to Quantify Cardiac Biomarkers in Human Serum

For a biosensing platform to have clinical relevance in point-of-care (POC) settings, assay sensitivity, reproducibility, and ability to reliably monitor analytes against the background of human serum are crucial.
Nanoimprinting lithography (NIL) was used to fabricate, at a low cost, sensing areas as large as 1.5 mm x 1.5 mm. The sensing surface was made of high-fidelity arrays of nanoholes, each with an area of about 140 nm2. The great reproducibility of NIL made it possible to employ a one-chip, one-measurement strategy on 12 individually manufactured surfaces, with minimal chip-to-chip variation. These nanoimprinted localized surface plasmon resonance (LSPR) chips were extensively tested on their ability to reliably measure a bioanalyte at concentrations varying from 2.5 to 75 ng/mL amidst the background of a complex biofluid-in this case, human serum. The high fidelity of NIL enables the generation of large sensing areas, which in turn eliminates the need for a microscope, as this biosensor can be easily interfaced with a commonly available laboratory light source. These biosensors can detect cardiac troponin in serum with a high sensitivity, at a limit of detection (LOD) of 0.55 ng/mL, which is clinically relevant. They also show low chip-to-chip variance (due to the high quality of the fabrication process). The results are commensurable with widely used enzyme-linked immunosorbent assay (ELISA)-based assays, but the technique retains the advantages of an LSPR-based sensing platform (i.e., amenability to miniaturization and multiplexing, making it more feasible for POC applications).

This work describes a nanoimprinting lithography method to fabricate high-quality sensing arrays that work on the principle of extraordinary optical transmission. The biosensor is low-cost, robust, easy to use, and can detect cardiac troponin I in serum at clinically relevant concentrations (99th percentile cutoff &#8764;10-400 pg/mL, depending on the assay).

For a biosensing platform to have clinical relevance in point-of-care (POC) settings, assay sensitivity, reproducibility, and ability to reliably monitor ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

Time-Resolved In Vivo Measurement of Neuropeptide Dynamics by Capacitive Immunoprobe in Porcine Heart

The ability to measure biomarkers in vivo relevant to the assessment of disease progression is of great interest to the scientific and medical communities. The resolution of results obtained from current methods of measuring certain biomarkers can take several days or weeks to obtain, as they can be limited in resolution both spatially and temporally (e.g., fluid compartment microdialysis of interstitial fluid analyzed by enzyme-linked immunosorbent assay [ELISA], high-performance liquid chromatography [HPLC], or mass spectrometry); thus, their guidance of timely diagnosis and treatment is disrupted. In the present study, a unique technique for detecting and measuring peptide transmitters in vivo through the use of a capacitive immunoprobe biosensor (CI probe) is reported. The fabrication protocol and in vitro characterization of these probes are described. Measurements of sympathetic stimulation-evoked neuropeptide Y (NPY) release in vivo are provided. NPY release is correlated to the sympathetic release of norepinephrine for reference. The data demonstrate an approach for the fast and localized measurement of neuropeptides in vivo. Future applications include intraoperative real-time assessment of disease progression and minimally invasive catheter-based deployment of these probes.

Time-Resolved In Vivo Measurement of Neuropeptide Dynamics by Capacitive Immunoprobe in Porcine Heart

Established immunochemical methods to measure peptide transmitters in vivo rely on microdialysis or bulk fluid draw to obtain the sample for offline analysis. However, these suffer from spatiotemporal limitations. The present protocol describes the fabrication and application of a capacitive immunoprobe biosensor that overcomes the limitations of the existing techniques.

The ability to measure biomarkers in vivo relevant to the assessment of disease progression is of great interest to the scientific and medical ...

High-Performance Graphene-Modified Sensing Chip for SARS-CoV-2 Detection

This sensing prototype model involves the development of a reusable, twofold graphene oxide (GrO)-glazed double inter-digitated capacitive (DIDC) detecting chip for detecting severe acute respiratory syndrome coronavirus 2 virus (SARS-CoV-2) specifically and rapidly. The fabricated DIDC comprises a Ti/Pt-containing glass substrate glazed with graphene oxide (GrO), which is further chemically modified with EDC-NHS to immobilize antibodies (Abs) hostile to SARS-CoV-2 based on the spike (S1) protein of the virus. The results of insightful investigations showed that GrO gave an ideal engineered surface for Ab immobilization and enhanced the capacitance to allow higher sensitivity and low sensing limits. These tunable elements helped accomplish a wide sensing range (1.0 mg/mL to 1.0 fg/mL), a minimum sensing limit of 1 fg/mL, high responsiveness and good linearity of 18.56 nF/g, and a fast reaction time of 3 s. Besides, in terms of developing financially viable point-of-care (POC) testing frameworks, the reusability of the GrO-DIDC biochip in this study is good. Significantly, the biochip is specific against blood-borne antigens and is stable for up to 10 days at 5 &#176;C. Due to its compactness, this scaled-down biosensor has the potential for POC diagnostics of COVID-19 infection. This system can also detect other severe viral diseases, although an approval step utilizing other virus examples is under development.

The present protocol describes the fabrication of low-cost biosensing prototypes based on useful nanosystems for accurately detecting viral proteins (at the Fg level). Such a tiny sensor platform allows for point-of-care applications that can be integrated with the Internet of Medical Things (IoMT) to meet telemedicine objectives.

Ultrasensitive Detection of Biomarkers by Using a Molecular Imprinting Based Capacitive Biosensor

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A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

ELIME (Enzyme Linked Immuno Magnetic Electrochemical) Method for Mycotoxin Detection

Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

Exploring Biomolecular Interaction Between the Molecular Chaperone Hsp90 and Its Client Protein Kinase Cdc37 using Field-Effect Biosensing Technology

The Use of a β-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions

Using Extraordinary Optical Transmission to Quantify Cardiac Biomarkers in Human Serum

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

Time-Resolved In Vivo Measurement of Neuropeptide Dynamics by Capacitive Immunoprobe in Porcine Heart

High-Performance Graphene-Modified Sensing Chip for SARS-CoV-2 Detection