The work reported herein was supported by grants from Auburn University AUDFS and USAF CRADA 07-277-60MDG-01. The views expressed in this article are those of the authors, and do not reflect the official policy or position of the United States Air Force, Department of Defense, or the U.S. Government.

1. Setting the Stage
<ol>
	<li>Obtain type strain S. aureus ATCC 12600, S. aureus ATCC 27690 and Bacillus subtilis ATCC 6051. Methicillin-resistant strains of S. aureus - MRSA1, MRSA 2, MRSA 5, MRSA 13, MRSA 26, MRSA 34, MRSA 45, B. anthracis Sterne, Salmonella typhimurium LT2, Shigella flexneri, Yersinia enterocolotica, Proteus mirabilis, Klebsiella pneumoniae 13882; The lytic phage 12600.</li>
	<li>Obtain PBP 2a antibody conjugated latex beads.</li>
	<li>Prepare NZY medium as described 21.</li>
	<li>Obtain phosphate buffered saline solution (PBS).</li>
	<li>Prepare deionized water as subphase for Langmuir-Blodgett (LB) monolayer deposition.</li>
</ol>
2. Bacteriophage Propagation and Titration
<ol>
	<li>Incubate 5 ml of overnight culture of S. aureus ATCC 12600 (3.6 x 108 colony forming units (CFU)/ml) with 500 &mu;l of phage (3 x 109 PFU/ml) in 500 ml NZY medium in 2 L flack on shaker-incubator at 37 &deg;C overnight.</li>
	<li>Centrifuge culture at 4,424 x g for 10 min at 4 &deg;C.</li>
	<li>Re-centrifuged supernatant at 11,325 x g for 10 min at 4 &deg;C.</li>
	<li>Filter supernatant through a 0.22 &mu;m Millipore filter.</li>
	<li>Centrifuge filtrate at 75,395 x g for 1.5 hr.</li>
	<li>Dissolve pellet in 100 &mu;l of distilled water.</li>
	<li>Prepare 10-fold dilutions of phage suspension in NZY medium.</li>
	<li>Plate 1 ml of overnight (ON) culture of S. aureus ATCC 12600 onto each of 2 plates with NZY agar to make sure all surface is covered. Remove the excess of culture and let the surface dry for 30 min.</li>
	<li>Spot a sample (10 &mu;l) of appropriate phage dilution onto the surface of the plate and incubate at 37 oC for 18-24 hr.</li>
	<li>Examine the formation of plaques and calculate the phage titer. Bacteriophage titers (T) are estimated on the basis of the number of plaques (N) formed per 10 &mu;l volume (v) of phage suspension and the dilution factor (F). The titer was calculated by formula: T=N/(vxF). For example, for the number of plaques 75 at the dilution 10-7 and the volume 10 &mu;l (10 x 10-3 ml), the titer is equal 75 / (10 x 10-3 x 10-7) = 7.5 x 1010 plaque forming units (PFU) per ml of suspension.</li>
</ol>
3. Gold-immobilized Phage
<ol>
	<li>Clean gold-coated quartz pieces (60 mm2) by plasma etching in argon for 10 min and then sterilize for 6 hr under UV light in a sterile cabinet.</li>
	<li>Add 50 microliters of 1 x 109 PFU/ml phage suspension to the gold surface of each piece in a sterile Petri dish, and then incubate overnight in a humid chamber at room temperature.</li>
	<li>Remove and titer the remaining phage suspension.</li>
	<li>Wash pieces with bound phage 5 times with PBS to remove unbound phage.</li>
</ol>
4. Testing of Immobilized Phage Infectivity on a Dry Surface
<ol>
	<li>Spread an O/N culture of S. aureus ATCC 12600 onto an NZY agar plate and allow drying.</li>
	<li>Place a gold piece with immobilized phage onto the plate face down.</li>
	<li>Observe a zone of lysis after 12 hr incubation at 37 &deg;C that indicates infectivity.</li>
	<li>Use gold covered pieces with no phage in control experiments.</li>
</ol>
5. Testing of Lytic Activity of Free and Bound Phage in Liquid
<ol>
	<li>Add an ON culture of S. aureus ATCC 12600 to 10 ml of NZY in every of four 300 ml sidearm flasks (6 x 106 CFU/ml in each flask).</li>
	<li>Add In two flasks 2 x 106 PFU/ml of free phage.</li>
	<li>Use the other two flasks as controls.</li>
	<li>Monitor the time course of S. aureus cell lysis for 570 min by optical density measurement (OD600) at 30 min intervals for all flasks.</li>
	<li>Take a final measurement at 24 hr.</li>
	<li>Repeat steps 5.1-5.5, but use gold pieces with bound phages instead of free phages, and gold pieces with no phage in control flasks.</li>
</ol>
6. Loss of Bound Phages During Incubation
<ol>
	<li>Add 50 &mu;l of 1 x 109 PFU/ml phage onto the surface of sterile gold piece in a Petri dish that is placed into a humid chamber overnight at room temperature.</li>
	<li>Remove the suspension with unbound phage from the gold surface and titer.</li>
	<li>Wash the gold piece with bound phage 5 times with PBS and place in 1 ml NZY solution in 15 ml tube in shaker-incubator at 37 &deg;C for 8 hr.</li>
	<li>Determine the titer of the detached phage as described in 2.</li>
</ol>
7. Phage Spheroids Preparation
<ol>
	<li>Combining 400 &mu;l of stock phage 12600 suspension (1011 PFU/ml) with an equal volume of spectrophotometric grade chloroform in 1 ml vial at room temperature.</li>
	<li>Gently vortex the phage-chloroform suspension 5-6 times (5 sec intervals) over one minute duration.</li>
	<li>Allowed the suspension to stabilize for 30 sec and then pipette of the top phase containing spheroids was pipetted off for monolayer preparation.</li>
	<li>Take a few microliters of the spheroids suspension for the transmission and scanning electron microscopy.</li>
	<li>Use a remaining spheroid suspension for biosensor manufacturing.</li>
</ol>
8. Biosensor Preparation
<ol>
	<li>Clean QCM-D sensors by plasma etching in argon for 10 min PDC-32G Harrick plasma cleaner.</li>
	<li>Rinse sensor with hexane to remove any organic impurities.</li>
	<li>Prepare and clean a trough of the film balance as described in 22.</li>
	<li>Fill LB trough with a subphase solution and clean it with a trough barrier and stabilize the trough at 20&plusmn;0.1 &deg;C</li>
	<li>Spread 300 &mu;l aliquot of phage 12600 in aqueous suspension (1011 PFU/ml) onto LB subphase.</li>
	<li>Prepare phage monolayer onto LB subphase solution by allowing 300 &mu;l aliquot of phage 12600 aqueous suspension (1011 PFU/ml) to run down an inclined wettable glass rod that is partially submersed into the subphase 22,23.</li>
	<li>Stabilize the prepared monolayer for 10 min, and then compress it at a rate of 30 mm/min (45 cm2/min), until a constant pressure 19 N/m is attained.</li>
	<li>Perform a vertical film deposition onto perpendicular positioned QCM-D sensor at a rate of 4.5 mm/min by successively dipping sensor in and out of the monolayer seven times. Electron microscopy of deposited phage monolayers before exposure to bacterial samples showed that the layers were continuous, homogeneous, and uniform (data not shown).</li>
	<li>Repeat steps 4.5-4.8 with a phage spheroid suspension that is prepared in 3.</li>
	<li>Use fabricated phage biosensors for MRSA detection, electron microscopy, and ellipsometry.</li>
</ol>
9. Biosensor Testing, Discrimination of Methicillin Resistant and Sensitive Staphylococcus Bacteria
<ol>
	<li>In this protocol, biosensors with unmodified and modified phage and phage spheroids are prepared. A quartz crystal microbalance with four sensor flow chambers is used to monitor binding of bacteria to the phage immobilized on QCM sensor surface. PBP 2a antibody conjugated latex beads are utilized to discriminate between methicillin resistant (MRSA) and sensitive staphylococcus bacteria. All measurements are conducted in the flow mode (50 &mu;l/min) at 5 MHz.</li>
	<li>Establish a base line resonance frequency of the QCM sensor in water (~30 min).</li>
	<li>Draw a suspension of live methicillin sensitive staphylococcus bacterial cells in water (109 CFU/ml) through the test cell.</li>
	<li>Continuously monitor changes in the sensors resonance frequency and energy dissipation for the first overtone, using the Q-Soft software.</li>
	<li>When the changes in the sensors resonance frequency and dissipation reached saturation levels, add the suspension of PBP antibody conjugated latex beads (6.77 x1010 beads/ml) to the flow during the continuous monitoring of frequency and dissipation.</li>
	<li>Repeat steps 9.2-9.5 for methicillin resistant staphylococcus bacterial cells (MRSA).</li>
	<li>Define a frequency change through the mass change due to bacteria binding by the Sauerbrey equation 24,25 &Delta;f=-&Delta;m xn/C, where C is the mass sensitivity constant (C=17.7 ng x cm-2 x Hz-1 at 5 MHz), m is mass and n is the overtone number (n = 1).</li>
	<li>Define an energy dissipation change (&Delta;D) from the recording the exponential decay of oscillation (frequency and amplitude dampening, which allowed quantification of the energy dissipated and stored during one period of oscillation, Edissipated and Estored, respectively: &Delta;D = Edissipated / 2&pi;Estored. &Delta;D is measured in dissipation units (DU), one DU = 10-6 relative units).</li>
</ol>

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The authors declare that they have no competing financial interests.

It is well known that phages can be used as biosensor probes for bacterial pathogens 28. It is demonstrated in this work that phage together with PBP 2a antibodies can be utilized to resolve the old problem: rapid discrimination antibiotic resistant and sensitive strains.
It was found however those normal unmodified staphylococcal phages are not suitable for bacteria detection with QCM devices, even though they bind bacteria. The phage tail is so long that acoustic waves cannot "rea...

Methicillin resistant strains of Staphylococcus aureus have been suggested as a factor in essential infections and nosocomial outbreaks 4-8. Common ways of the recognition of methicillin resistance, such as the disk diffusion oxacillin agar screen test, or broth microdilution, rely on tailored culture conditions to enhance the expression of resistance. Alterations include the utilization of oxacillin, incubation at 30 or 35 &deg;C rather than 37 &deg;C, and the inclusion of NaCl to the growth medium. Furthermore, for correct detection by these types of techniques, a long incubation period of 24 hr instead of 16 to 18 hr is required. Rapid techniques with appropriate (&gt;96%) level of sensitivity for identification of methicillin resistance include automated microdilution techniques such as the Vitek GPS-SA card, the Rapid ATB Staph system, and the Rapid Microscan Panel system which produce results after 3-11 hr 9-11. The Crystal MRSA ID system is a rapid method based upon recognition of growth of S. aureus in the presence of 2% NaCl and 4 mg of oxacillin per liter with an oxygen-sensitive fluorescence sensor. Claimed sensitivities range between 91 to 100% after 4 hr of incubation 12-14. These phenotypic methods are limited in their accuracies by the impact of prevalent strains that express heterogeneous resistance. Therefore, the best widely accepted methods for the recognition of methicillin resistance is PCR or DNA hybridization of the mecA gene 15. However this technique requires purified DNA and is extremely sensitive to various admixtures (impurities), which include cell debris16.
Furthermore, these techniques need a long time to perform. Strategies to the recognition of the mecA gene product, protein PBP 2a, could be utilized to determine resistance and may be more reliable compared to standard test techniques 17.
It had been earlier shown that bacteriophage 12600 can be utilized as a recognition probe for Staphylococcus aureus strains including those having methicillin resistance 1,2,18. In this work we proposed a novel technique in the specific recognition and detection of MRSA, such as the recognition of bacteria along with conformation of MRSA in real time. For this specific purpose a S. aureus bacteriophage with a wide spectrum of hosts (including MRSA strains) combined with monoclonal antibody against protein (PBP 2a) have been used. PBP 2a is a cell wall protein and it is the cause of antibiotic resistivity of MRSA. However PBP 2a antibody is not specific for S. aureus since some other bacteria have antibiotic binding proteins with sequence similarity to PBP 2a 19,20. Consequently in this work, S. aureus bacteriophage and antibodies against PBP 2a protein have been used. To be able to develop a biosensor to specifically detect and identify MRSA a device with a two-step action has been utilized. The initial step used a S. aureus bacteriophage monolayer as a sensor probe, while the second step employed PBP 2a specific antibodies. Therefore, step one will recognize S. aureus bacteria, as the other one will be sensitive to the antibiotic-binding protein. When signals received from two steps are positive, it indicates the specific detection of MRSA.

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagents</td>
		 </tr>
		 <tr>
			<td>Phosphate buffered saline (PBS) </td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>P4417 </td>
			<td></td>
		 </tr>
		 <tr>
			<td>spectrophotometric-grade chloroform </td>
			<td>Sigma-Aldrich, 
				St. Louis, MO </td>
			<td>154733 </td>
			<td>(99.8% A.C.S.) </td>
		 </tr>
		 <tr>
			<td>Hexane-Anhydrous </td>
			<td>Sigma-Aldrich, 
				St. Louis, MO</td>
			<td>29609-0</td>
			<td>(95%)</td>
		 </tr>
		 <tr>
			<td>Ethyl Alcohol </td>
			<td>Pharmco products Inc. Brookfield, CT </td>
			<td>64-17-5</td>
			<td>190 Proof</td>
		 </tr>	
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		 </tr>
		 <tr>
			<td>PBP 2a antibody conjugated latex beads</td>
			<td>Denka Seiken Co., Ltd, Tokyo, Japan</td>
			<td>The MRSA-Screen test</td>
			<td></td>
		 </tr>
		 <tr>
			<td>S. aureus ATCC 12600, S. aureus ATCC 27690 and Bacillus subtilis ATCC 6051 from </td>
			<td>American Type Culture Collection (Manassas, VA);</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>MRSA1, MRSA 2, MRSA 5, MRSA 13, MRSA 26, MRSA 34, MRSA 45, B. anthracis Sterne, Salmonella typhimurium LT2, Shigella flexneri, Yersinia enterocolotica, Proteus mirabilis, Klebsiella pneumoniae 13882; The lytic phage 12600 </td>
			<td>The culture collection of Auburn University, Auburn, AL</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Centrifuge </td>
			<td>Beckman Coulter</td>
			<td>Optima L-90K
				Ultra Centrifuge</td>
			<td></td>
		 </tr>
		 <tr>
			<td>KSV 2200 LB film balance</td>
			<td>KSV Chemicals, Finland</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Light microscope optical system </td>
			<td>CitoViva Technology Inc., Auburn, AL </td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>QCM-D</td>
			<td>Q-Sense AB, V&auml;stra Fr&ouml;lunda, Sweden </td>
			<td>E4</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Scanning electron microscope (SEM)</td>
			<td>JEOL USA Inc., Peabody, MA</td>
			<td>JEOL-7000F SEM </td>
			<td></td>
		 </tr>
		 <tr>
			<td>Transmitting electron microscopy (TEM)</td>
			<td>JEOL USA Inc., Peabody, MA</td>
			<td>JEOL, JEM 2010 </td>
			<td></td>
		 </tr>
		 <tr>
			<td>Stericup, Presterilized</td>
			<td>Millipore Corporation, Billerica, MA</td>
			<td>SCGPU05RE</td>
			<td>0.22 &mu;m, GP Express PLUS membrane</td>
		 </tr>
		 <tr>
			<td>Bio-Assay dish</td>
			<td>NUNC A/S, Denmark</td>
			<td>240835</td>
			<td>Dimensions(mm), 245 x 245 x 25</td>
		 </tr>
		 <tr>
			<td>Pipettes</td>
			<td>Gilson, Pipetman, France</td>
			<td>P100, P200, P1000</td>
			<td></td>
		 </tr>
		 <tr>
			<td>C24 Incubator Shaker</td>
			<td>New Brunswick Scientific, CT</td>
			<td>Classic C24</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Gold-coated quartz pieces</td>
			<td>Auburn University, AL</td>
			<td></td>
			<td>Homemade</td>
		 </tr>
		 <tr>
			<td>Petri dishes</td>
			<td>Fisher Brand, USA</td>
			<td>0875713</td>
			<td>100 mmX15 mm </td>
		 </tr>
		 <tr>
			<td>SterilGard III Advance</td>
			<td>The Baker Company, ME</td>
			<td>SG403</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Culture Growing Flasks</td>
			<td>Corning Incorporated, NY</td>
			<td>4995</td>
			<td>PYREX 250 ml Erlenmeyer flasks </td>
		 </tr>
		 <tr>
			<td>Optical Spectrometer</td>
			<td>Genesys 20. Thermo Spectronic, USA.</td>
			<td>4001</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Plasma Cleaner</td>
			<td>Harrick Plasma, USA</td>
			<td>PDC-32G</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Millipore water purification system</td>
			<td>Millipore</td>
			<td>Direct-Q</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Imaging Ellipsometer</td>
			<td>Accurion, USA</td>
			<td>nanofilm_ep3se
			</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Software </td>
			<td>Q-Sense AB, Sweden</td>
			<td>QSoft, QTools</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

biosensor for detection of antibiotic resistant staphylococcus bacteria

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) antibody conjugated latex beads have been utilized to create a biosensor designed for discrimination of methicillin resistant (MRSA) and sensitive (MSSA) S. aureus species 1,2. The lytic phages have been converted into phage spheroids by contact with water-chloroform interface. Phage spheroid monolayers have been moved onto a biosensor surface by Langmuir-Blodgett (LB) technique 3. The created biosensors have been examined by a quartz crystal microbalance with dissipation tracking (QCM-D) to evaluate bacteria-phage interactions. Bacteria-spheroid interactions led to reduced resonance frequency and a rise in dissipation energy for both MRSA and MSSA strains. After the bacterial binding, these sensors have been further exposed to the penicillin-binding protein antibody latex beads. Sensors analyzed with MRSA responded to PBP 2a antibody beads; although sensors inspected with MSSA gave no response. This experimental distinction determines an unambiguous discrimination between methicillin resistant and sensitive S. aureus strains. Equally bound and unbound bacteriophages suppress bacterial growth on surfaces and in water suspensions. Once lytic phages are changed into spheroids, they retain their strong lytic activity and show high bacterial capture capability. The phage and phage spheroids can be utilized for testing and sterilization of antibiotic resistant microorganisms. Other applications may include use in bacteriophage therapy and antimicrobial surfaces.

The phage demonstrated lytic activity against all tested strains of S. aureus, including MRSA strains, as indicated by the phage spot test. Plaque sizes generally ranged from 5 to 15 mm. No activity was found against other test-cultures (Table 1).
A normal growth of S. aureus ATCC 12600 in NZY medium on shaker-incubator at 37 &deg;C is shown in Figure 1A (a curve labeled by empty circles). The number of bacteria increased from 3.2 x 106<...

Watch this Scientific Journal Video about Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria at JoVE.com

Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

Lytic phage biosensors and antibody beads are able to discriminate between methicillin resistant (MRSA) and sensitive staphylococcus bacteria. The phages were immobilized by a Langmuir-Blodgett method onto a surface of a quartz crystal microbalance sensor and worked as broad range staphylococcus probes. Antibody beads recognize MRSA.

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) ...

biosensor-for-detection-antibiotic-resistant-staphylococcus

Research

JoVE Journal

Bioengineering

24.0K Views.  Auburn University. Lytic phage biosensors and antibody beads are able to discriminate between methicillin resistant (MRSA) and sensitive staphylococcus bacteria. The phages were immobilized by a Langmuir-Blodgett method onto a surface of a quartz crystal microbalance sensor and worked as broad range staphylococcus probes. Antibody beads recognize MRSA.

Video: Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Fabrication of Carbon Nanotube High-Frequency Nanoelectronic Biosensor for Sensing in High Ionic Strength Solutions

The unique electronic properties and high surface-to-volume ratios of single-walled carbon nanotubes (SWNT) and semiconductor nanowires (NW) 1-4 make them good candidates for high sensitivity biosensors. When a charged molecule binds to such a sensor surface, it alters the carrier density5 in the sensor, resulting in changes in its DC conductance. However, in an ionic solution a charged surface also attracts counter-ions from the solution, forming an electrical double layer (EDL). This EDL effectively screens off the charge, and in physiologically relevant conditions ~100 millimolar (mM), the characteristic charge screening length (Debye length) is less than a nanometer (nm). Thus, in high ionic strength solutions, charge based (DC) detection is fundamentally impeded6-8.
We overcome charge screening effects by detecting molecular dipoles rather than charges at high frequency, by operating carbon nanotube field effect transistors as high frequency mixers9-11. At high frequencies, the AC drive force can no longer overcome the solution drag and the ions in solution do not have sufficient time to form the EDL. Further, frequency mixing technique allows us to operate at frequencies high enough to overcome ionic screening, and yet detect the sensing signals at lower frequencies11-12. Also, the high transconductance of SWNT transistors provides an internal gain for the sensing signal, which obviates the need for external signal amplifier.
Here, we describe the protocol to (a) fabricate SWNT transistors, (b) functionalize biomolecules to the nanotube13, (c) design and stamp a poly-dimethylsiloxane (PDMS) micro-fluidic chamber14 onto the device, and (d) carry out high frequency sensing in different ionic strength solutions11.

We describe the device fabrication and measurement protocol for carbon nanotube based high frequency biosensors. The high frequency sensing technique mitigates the fundamental ionic (Debye) screening effect and allows nanotube biosensor to be operated in high ionic strength solutions where conventional electronic biosensors fail. Our technology provides a unique platform for point-of-care (POC) electronic biosensors operating in physiologically relevant conditions.

The unique electronic properties and high surface-to-volume ratios of single-walled carbon nanotubes (SWNT) and semiconductor nanowires (NW) 1-4 make them ...

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 ...

Stress-induced Antibiotic Susceptibility Testing on a Chip

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria survive these stressful conditions. The hypothesis behind this method is new: stress activation of biochemical pathways, which are targets of antibiotics, can accelerate antibiotic susceptibility testing. As compared to standard antibiotic susceptibility testing methods, the rate-limiting step -&#160;bacterial growth -&#160;is omitted during antibiotic application. The technical implementation of the method is in a combination of standard techniques and innovative approaches. The standard parts of the method include bacterial culture protocols, defining microfluidic channels in polydimethylsiloxane&#160;(PDMS), cell viability monitoring with fluorescence, and batch image processing for bacteria counting. Innovative parts of the method are in the use of culture media flow for mechanical stress application, use of enzymes to damage but not kill the bacteria, and use of microarray substrates for bacterial attachment. The developed platform can be used in antibiotic and nonantibiotic related drug development and testing. As compared to the standard bacterial suspension experiments, the effect of the drug can be turned on and off repeatedly over controlled time periods. Repetitive observation of the same bacterial population is possible over the course of the same experiment.

We have developed a microfluidic platform for rapid antibiotic susceptibility testing. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria can survive these stressful conditions.

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over ...

Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and progression of pathology in several neurodegenerative diseases, including Alzheimer's disease and the related tauopathies. The potentially critical role of tau seeds in disease progression strongly supports the need for a sensitive assay that readily detects seeding activity in biological samples. 
By combining the specificity of fluorescence resonance energy transfer (FRET), the sensitivity of flow cytometry, and the stability of a monoclonal cell line, an ultra-sensitive seeding assay has been engineered and is compatible with seed detection from recombinant or biological samples, including human and mouse brain homogenates. The assay employs monoclonal HEK 293T cells that stably express the aggregation-prone repeat domain (RD) of tau harboring the disease-associated P301S mutation fused to either CFP or YFP, which produce a FRET signal upon protein aggregation. The uptake of proteopathic tau seeds (but not other proteins) into the biosensor cells stimulates aggregation of RD-CFP and RD-YFP, and flow cytometry sensitively and quantitatively monitors this aggregation-induced FRET. The assay detects femtomolar concentrations (monomer equivalent) of recombinant tau seeds, has a dynamic range spanning three orders of magnitude, and is compatible with brain homogenates from tauopathy transgenic mice and human tauopathy subjects. With slight modifications, the assay can also detect seeding activity of other proteopathic seeds, such as &#945;-synuclein, and is also compatible with primary neuronal cultures. The ease, sensitivity, and broad applicability of FRET flow cytometry makes it useful to study a wide range of protein aggregation disorders.

Cell-to-cell transfer of protein aggregates, or proteopathic seeds, may underlie the progression of pathology in neurodegenerative diseases. Here, a novel FRET flow cytometry assay is described that enables specific and sensitive detection of seeding activity from recombinant or biological samples.

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and ...

High-throughput Identification of Bacteria Repellent Polymers for Medical Devices

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as contributing to antimicrobial resistance. One possible avenue for the reduction of device-associated infections is the identification of bacteria-repellent polymer coatings for these devices, which would prevent bacterial binding at the initial attachment step. A method for the identification of such repellent polymers, based on the parallel screening of hundreds of polymers using a microarray, is described here. This high-throughput method resulted in the identification of a range of promising polymers that resisted binding of various clinically relevant bacterial species individually and also as multi-species communities. One polymer, PA13 (poly(methylmethacrylate-co-dimethylacrylamide)), demonstrated significant reduction in attachment of a number of hospital isolates when coated onto two commercially available central venous catheters. The method described could be applied to identify polymers for a wide range of applications in which modification of bacterial attachment is important.

A high-throughput microarray method for the identification of polymers which reduce bacterial surface binding on medical devices is described.

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

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 ...

Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, significantly affecting the rate of human mortality and morbidity. Conventional methods for the detection of V. parahaemolyticus such as culture-based methods, immunological assays, and molecular-based methods require complicated sample handling and are time-consuming, tedious, and costly. Recently, biosensors have proven to be a promising and comprehensive detection method with the advantages of fast detection, cost-effectiveness, and practicality. This research focuses on developing a rapid method of detecting V. parahaemolyticus with high selectivity and sensitivity using the principles of DNA hybridization. In the work, characterization of synthesized polylactic acid-stabilized gold nanoparticles (PLA-AuNPs) was achieved using X-ray Diffraction (XRD), Ultraviolet-visible Spectroscopy (UV-Vis), Transmission Electron Microscopy (TEM), Field-emission Scanning Electron Microscopy (FESEM), and Cyclic Voltammetry (CV). We also carried out further testing of stability, sensitivity, and reproducibility of the PLA-AuNPs. We found that the PLA-AuNPs formed a sound structure of stabilized nanoparticles in aqueous solution. We also observed that the sensitivity improved as a result of the smaller charge transfer resistance (Rct) value and an increase of active surface area (0.41 cm2). The development of our DNA biosensor was based on modification of a screen-printed carbon electrode (SPCE) with PLA-AuNPs and using methylene blue (MB) as the redox indicator. We assessed the immobilization and hybridization events by differential pulse voltammetry (DPV). We found that complementary, non-complementary, and mismatched oligonucleotides were specifically distinguished by the fabricated biosensor. It also showed reliably sensitive detection in cross-reactivity studies against various food-borne pathogens and in the identification of V. parahaemolyticus in fresh cockles.

A protocol for the development of an electrochemical DNA biosensor comprising a polylactic acid-stabilized, gold nanoparticles-modified, screen-printed carbon electrode to detect Vibrio parahaemolyticus is presented.

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, ...

Visual Detection of Multiple Nucleic Acids in a Capillary Array

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently needed in disease diagnosis, microbial monitoring, genetically modified organism (GMO) detection, and forensic analysis. We have previously described the platform called CALM (Capillary Array-based Loop-mediated isothermal amplification for Multiplex visual detection of nucleic acids). Herein, we describe improved fabrication and performance processes for this platform. Here, we apply a small, ready-to-use cassette assembled by capillary array for multiplex visual detection of nucleic acids. The capillary array is pre-treated into a hydrophobic and hydrophilic pattern before fixing loop-mediated isothermal amplification (LAMP) primer sets in capillaries. After assembly of the loading adaptor, LAMP reaction mixture is loaded and isolated into each capillary, due to capillary force by a single pipetting step. The LAMP reactions are performed in parallel in the capillaries. The results are visually read out by illumination with a hand-held UV flashlight. Using this platform, we demonstrate monitoring of 8 frequently appearing elements and genes in GMO samples with high specificity and sensitivity. In summary, the platform described herein is intended to facilitate the detection of multiple nucleic acids. We believe it will be widely applicable in fields where high-throughput nucleic acid analysis is required.

This protocol describes the fabrication of a small, ready-to-use cassette that can be applied for visual detection of multiple nucleic acids in a single, test that is easy to operate. In this approach, a capillary array was used for multiplex and highly efficient detection of GMO targets.

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently ...

Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

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Rozdziały w tym wideo

Fluorescence detection methods for microfluidic droplet platforms

Fabrication of Carbon Nanotube High-Frequency Nanoelectronic Biosensor for Sensing in High Ionic Strength Solutions

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

Stress-induced Antibiotic Susceptibility Testing on a Chip

Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry

High-throughput Identification of Bacteria Repellent Polymers for Medical Devices

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

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

Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

Visual Detection of Multiple Nucleic Acids in a Capillary Array