Sandia is multiprogram laboratory operated by Sandia Corporation, a Lockheed Martin Company, for the United Stated Department of Energy's National Nuclear Security Administration under Contract DE-AC04-94AL85000. The authors acknowledge funding from Sandia National Laboratories' Laboratory Directed Research &amp; Development (LDRD) program.

1. Microneedle Fabrication
<ol>
	<li>Using three-dimensional modeling software Solidworks (Dassault Systemes SA, Velizy, France), design a pyramidal-shaped hollow microneedle array (Figure 1).3-5</li>
	<li>Design a support structure for the microneedle array using Magics RP 13 software (Materialise NV, Leuven, Belgium). The support structure allows the resin to drain from the device during fabrication and provides a base on which the microneedles are built. An example support structure is shown in Figure 1.</li>
	<li>The linked support and microneedle array files are uploaded into the Perfactory RP software (EnvisionTEC GmbH, Gladbeck, Germany), which controls the fabrication process. Within this software package, choose the number of microneedle arrays to be fabricated and determine the placement of devices on the fabrication plate.</li>
	<li>Run calibration in ultraviolet mode at 180 mW for the Perfactory rapid prototyping manufacturing system and verify the deviation in energy is within &plusmn;2 mW.</li>
	<li>Once fabrication is complete, remove the microneedle arrays from the baseplate and develop in isopropanol for 15 minutes. Dry the arrays with compressed air and cure the microneedles at room temperature for 50 seconds in the Otoflash Postcuring System (EnvisionTEC GmbH, Gladbeck, Germany) to ensure complete polymerization.</li>
	<li>Validate microneedle fabrication via microscopy and verify that each microneedle bore is hollow and unobstructed. Fully-fabricated microneedles are shown in Figure 2.</li>
</ol>
2. Fabrication of Carbon Paste Electrode Arrays
<ol>
	<li>Use a 60 W Model 6.75 CO2 raster/vector laser system (Universal Laser Systems, Inc., Scottsdale, AZ) to cut holes and expose the underlying individually addressable connecting copper wires in a flat flexible cable (21039-0249), which was obtained from a commercial source (Molex Connector Corp., Lisle, IL) (Figure 3 (A and B)). Place the flat flexible cables in a jig to properly align them on the laser ablation plate. Use a rastering approach to create 500 &mu;m diameter cavities in the insulating portion of the flexible cable. Patterns for ablation are created in CorelDraw (Corel, Ottawa, Ontario) and sent to the laser system.</li>
	<li>Clean the modified flat flexible cables with an airbrush that sprays acetone at 40 psi. Finish cleaning them by rinsing with isopropanol and deionized water. Verify under a microscope that no insulating film remains over the exposed copper strips.</li>
	<li>The next step is to create a holding cavity for packing of carbon pastes. Melinex tape (0.002" thickness coated on a single side with pressure sensitive acrylic adhesive) is ablated with the same pattern as the electrode strips, oriented over the ablated electrode strips, and compressed at 3000 psi for 2 minutes to ensure a proper connection. In this case, the cavity diameter is 750 &mu;m.</li>
	<li>An additional layer of Melinex tape (0.004" thickness coated on two sides with pressure sensitive acrylic adhesive) is subsequently ablated in the same pattern as the single-sided adhesive tape and is used after alignment to bond the microneedle arrays to the carbon paste electrode arrays.</li>
</ol>
3. Synthesis of Functional Carbon Pastes and Packing of Electrode Cavities
<ol>
	<li>The glucose sensitive carbon paste is based off of a previous recipe and is obtained by mixing 10 mg of glucose oxidase and 2.2 mg of poly(ethylenimine) until a homogeneous mixture is obtained.16 To this mixture, 60 mg of rhodium on carbon powder (5% loading) is added. 40 mg mineral oil is added and subsequently mixed. The pastes are stored at 4 &deg;C until use; the pastes are used up to one week after preparation.</li>
	<li>The pH sensitive carbon paste is obtained by mixing 30% (w/w) mineral oil and 70% (w/w) graphite powder. Pack paste into the electrode cavity as described in section 3.4. Make solution of 10 mM Fast Blue RR diazonium salt (4-benzoylamino-2,5-dimethoxybenzenediazonium chloride hemi (zinc chloride) salt) in 0.5 M phosphoric acid.17, Place a 20 &mu;l drop of this solution over the packed paste electrode for 30 minutes to spontaneously chemisorb the Fast Blue PR diazonium salt. Rinse with deionized water and store in buffer or deionized water when not in use.</li>
	<li>The lactate sensitive carbon paste is based off of a previous recipe and is obtained by mixing 2.5 mg of rhodium on carbon powder and 2.5 mg of lactate oxidase, alternating between 5 minutes of sonication and 5 minutes of vortexing for five rotations.18</li>
	<li>Packing of the modified pastes into the prepared flat flexible cable is accomplished by applying the respective pastes over the electrode cavities. Using a thin piece of plastic (e.g., an edge of a plastic weigh boat) as a trowel and pack the paste until a smooth surface is achieved. Repeat with a second clean weighing boat until excess paste is removed. Wash with deionized water. A schematic showing laser ablation to create cavities, packing of carbon pastes, and microneedle integration (described in section 2 and 3) is presented in Figure 3.</li>
</ol>
4. Detection and sensor calibration
<ol>
	<li>Lactate detection is accomplished by measuring the chronoamperometric response of the sensor at -0.15 V and recording the current after 15 seconds in 0.1 M phosphate buffer (pH=7.5). Figure 4(a) contains a schematic of the electrocatalytic reaction for detection of lactate.</li>
	<li>Glucose detection is performed in a similar manner by measuring the chronoamperometric response of the sensor at -0.05 V and recording the current after 15 seconds in 0.1 M phosphate buffer (pH 7.0). Figure 4(b) contains a schematic of the electrocatalytic reaction for detection of glucose.</li>
	<li>pH is monitored by running cyclic voltammetric scans from -0.7 V to 0.8 V at 100 mV/s and recording the position of the oxidative peak potential. A schematic of the redox reactions for pH detection is shown in Figure 5.</li>
	<li>Calibration curves for glucose and lactate sensors can be created by successive additions of the respective analyte; chronoamperometric measurements are performing after each analyte addition as described in sections 5.1 and 5.2. Alternatively, fixed potential chronoamperometric measurements can be made under stirring while allowing sufficient time (~10-100 seconds) in between each analyte addition for current stabilization.</li>
	<li>pH calibration curves can be created by measuring the position of the oxidative peak potential over a series of known pH values from 5 to 8 in 1.0 pH unit increments and recording cyclic voltammograms as described in section 5.3.</li>
</ol>
5. Representative Results
When obtaining chronoamperometric curves (e.g., for glucose detection or lactate detection) in quiescent solutions with modified carbon paste-filled microneedles, the current will immediately decrease upon application of the respective detection potential. It will eventually decay to a steady state value. A representative result is shown in Figure 6; this result was obtained from 2 mM additions of lactate and recording at the lactate microneedle. The solution must be briefly stirred after each lactate addition. The current after 15 seconds rises upon increasing the concentration of lactate; the current response can then be used to determine the concentration of lactate in an unknown solution. Alternatively, continuous monitoring can be used in a stirred solution (or in a flowing solution) as demonstrated for a solution with an increasing glucose concentration (Figure 5). Again, the increase in current upon increasing the concentration of glucose can be used to standardize the glucose response to an unknown solution. Sufficient time must be allowed after each spike in order to allow the solution to stabilize. Cyclic voltammograms at the pH sensitive microneedle in 0.1 M phosphate buffer are shown over four different pH solutions from 5 to 8 in 1 pH unit increments in Figure 6. The oxidative peak potential shifts with increasing pH; this phenomenon is used as an indicator of the pH value.
<img alt="Figure 1" src="/files/ftp_upload/4067/4067fig1.jpg" /> 
Figure 1. Images of the STL file of the microneedle array created in Solidworks (A) and of the print screen, which shows the support structure (B).
<img alt="Figure 2" src="/files/ftp_upload/4067/4067fig2.jpg" /> 
Figure 2. Scanning electron micrographs of the microneedle array (A) and a single microneedle within this array (B).
<img alt="Figure 3" src="/files/ftp_upload/4067/4067fig3.jpg" /> 
Figure 3. Schematic of flat flexible cable assembly. The steps involved include modifying the flat flexible cable (A), ablating the patterned circles (B), adding the initially ablated Melinex layer, which is filled with carbon paste (C), as well as adding the second ablated Melinex layer and mating the microneedle array (D). <a href="https://www.jove.com/files/ftp_upload/4067/4067fig3large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 4" src="/files/ftp_upload/4067/4067fig4.jpg" /> 
Figure 4. Calibration of lactate-sensitive paste with 15 second chronoamperometric scans at -0.15 V in 0.1 M phosphate buffer (pH=7.5). Each increase in current corresponds to a 2 mM addition of lactate.
<img alt="Figure 5" src="/files/ftp_upload/4067/4067fig5.jpg" /> 
Figure 5. Calibration of glucose-sensitive paste running chronoamperometric scans at -0.05 V. Each decrease in the response corresponds to a 2 mM addition of glucose. The 0.1 M phosphate buffer solution (pH=7.0) was stirred during calibration. External Ag/AgCl and Pt reference and counter electrodes were used in this study.
<img alt="Figure 6" src="/files/ftp_upload/4067/4067fig6.jpg" /> 
Figure 6. Cyclic voltammogram (CV) of pH sensitive carbon paste in 0.1 M phosphate buffer over pH 5-8 in 1 pH unit increments (teal=pH 8.0, green=pH 7.0, purple=pH 6.0, red=pH 5.0). A fifth CV was used for analysis versus Ag/AgCl reference and Pt wire counter electrodes.

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No conflicts of interest declared.

Multiple aspects of the design of this microneedle-based sensor were considered prior to device fabrication. In order to use this sensor for real-time detection, the response time of the sensor must be low; in this protocol, each tested sensor exhibited a response time below fifteen seconds. Pastes used in this protocol were also chosen for their selectivity within in vivo environments, which contain electroactive biomolecules that can interfere with electrode response. In addition to paste composition, th...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Flat flexible cable</td>
 <td>Molex</td>
 <td>3302/10SF</td>
 </tr>
 <tr>
 <td>0.003" Side sided tape</td>
 <td>Melinex</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>0.004" Double sided tape</td>
 <td>Melinex</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Lactate oxidase</td>
 <td>Sigma</td>
 <td>L0638</td>
 </tr>
 <tr>
 <td>Glucose oxidase</td>
 <td>Sigma</td>
 <td>G7141</td>
 </tr>
 <tr>
 <td>Rhodium on carbon</td>
 <td>Sigma</td>
 <td>206164</td>
 </tr>
 <tr>
 <td>Graphite powder</td>
 <td>Sigma</td>
 <td>385031000</td>
 </tr>
 <tr>
 <td>poly(ethylenimine)</td>
 <td>Acros</td>
 <td>178570010</td>
 </tr>
 <tr>
 <td>Mineral oil </td>
 <td>Sigma</td>
 <td>M5904</td>
 </tr>
 <tr>
 <td>Glucose</td>
 <td>Sigma</td>
 <td>G8270</td>
 </tr>
 <tr>
 <td>Lactate</td>
 <td>Sigma</td>
 <td>L1750</td>
 </tr>
 <tr>
 <td>Fast Blue RR salt</td>
 <td>Sigma</td>
 <td>F0500</td>
 </tr>
 <tr>
 <td>e-Shell 300</td>
 <td>EnvisionTEC</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>e-Shell 200</td>
 <td>EnvisionTEC</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ag/AgCl reference electrode</td>
 <td>Basi</td>
 <td>MF-2052</td>
 </tr>
 <tr>
 <td>Pt wire</td>
 <td>Basi</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PGSTAT12 AutolabPotentiostat </td>
 <td>EcoChemie</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Perfactory RP</td>
 <td>EnvisionTEC</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ottoflash Postcuring system</td>
 <td>EnvisionTEC</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Phosphoric acid</td>
 <td>Fisher</td>
 <td>A366-4</td>
 </tr>
 <tr>
 <td>60W Model 6.75 CO2 raster/vector laser system </td>
 <td>Universal Laser Systems</td>
 <td>PLS6.75</td>
 </tr>
 <tr>
 <td>CorelDraw</td>
 <td>Corel</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Solidworks </td>
 <td>Dassault Systemes</td>
 <td>2009</td>
 </tr>
 <tr>
 <td>Magics RP13</td>
 <td>Materialise </td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

hollow microneedle-based sensor for multiplexed transdermal electrochemical sensing

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals suffering from chronic medical conditions facile assessment of their immediate physiological state. Furthermore, it could serve as a research tool for analysis of complex, multifactorial medical conditions. In order for such a multianalyte sensor to be realized, it must be minimally invasive, sampling of interstitial fluid must occur without pain or harm to the user, and analysis must be rapid as well as selective.
Initially developed for pain-free drug delivery, microneedles have been used to deliver vaccines and pharmacologic agents (e.g., insulin) through the skin.1-2 Since these devices access the interstitial space, microneedles that are integrated with microelectrodes can be used as transdermal electrochemical sensors. Selective detection of glucose, glutamate, lactate, hydrogen peroxide, and ascorbic acid has been demonstrated using integrated microneedle-electrode devices with carbon fibers, modified carbon pastes, and platinum-coated polymer microneedles serving as transducing elements.3-7,8
This microneedle sensor technology has enabled a novel and sophisticated analytical approach for in situ and simultaneous detection of multiple analytes. Multiplexing offers the possibility of monitoring complex microenvironments, which are otherwise difficult to characterize in a rapid and minimally invasive manner. For example, this technology could be utilized for simultaneous monitoring of extracellular levels of, glucose, lactate and pH,9 which are important metabolic indicators of disease states7,10-14 (e.g., cancer proliferation) and exercise-induced acidosis.15

Watch this Scientific Journal Video about Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing at JoVE.com

Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

This article details the construction of a multiplexed microneedle-based sensor. The device is being developed for in situ sampling and electrochemical analysis of multiple analytes in a rapid and selective manner. We envision clinical medicine and biomedical research uses for these microneedle-based sensors.

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals ...

hollow-microneedle-based-sensor-for-multiplexed-transdermal

Research

JoVE Journal

Bioengineering

14.2K Views.  University of North Carolina and North Carolina State University. This article details the construction of a multiplexed microneedle-based sensor. The device is being developed for in situ sampling and electrochemical analysis of multiple analytes in a rapid and selective manner. We envision clinical medicine and biomedical research uses for these microneedle-based sensors.

Video: Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, environmental/agricultural/biodefense monitoring, nanobiotechnology, and more. For diagnostic applications, monitoring (detecting) the presence, absence, or abnormal expression of targeted proteomic or genomic biomarkers found in patient samples can be used to determine treatment approaches or therapy efficacy. In the research arena, information on molecular affinities and specificities are useful for fully characterizing the systems under investigation.
Many of the current systems employed to determine molecular concentrations or affinities rely on the use of labels. Examples of these systems include immunoassays such as the enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) techniques, gel electrophoresis assays, and mass spectrometry (MS). Generally, these labels are fluorescent, radiological, or colorimetric in nature and are directly or indirectly attached to the molecular target of interest. Though the use of labels is widely accepted and has some benefits, there are drawbacks which are stimulating the development of new label-free methods for measuring these interactions. These drawbacks include practical facets such as increased assay cost, reagent lifespan and usability, storage and safety concerns, wasted time and effort in labelling, and variability among the different reagents due to the labelling processes or labels themselves. On a scientific research basis, the use of these labels can also introduce difficulties such as concerns with effects on protein functionality/structure due to the presence of the attached labels and the inability to directly measure the interactions in real time.
Presented here is the use of a new label-free optical biosensor that is amenable to microarray studies, termed the Interferometric Reflectance Imaging Sensor (IRIS), for detecting proteins, DNA, antigenic material, whole pathogens (virions) and other biological material. The IRIS system has been demonstrated to have high sensitivity, precision, and reproducibility for different biomolecular interactions [1-3]. Benefits include multiplex imaging capacity, real time and endpoint measurement capabilities, and other high-throughput attributes such as reduced reagent consumption and a reduction in assay times. Additionally, the IRIS platform is simple to use, requires inexpensive equipment, and utilizes silicon-based solid phase assay components making it compatible with many contemporary surface chemistry approaches.
Here, we present the use of the IRIS system from preparation of probe arrays to incubation and measurement of target binding to analysis of the results in an endpoint format. The model system will be the capture of target antibodies which are specific for human serum albumin (HSA) on HSA-spotted substrates.

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor IRIS

Quantitative, high-throughput, real-time, and label-free biomolecular detection (DNA, protein, etc.) on SiO2 surfaces can be achieved using a simple interferometric technique which relies on LED illumination, minimal optical components, and a camera. The Interferometric Reflectance Imaging Sensor (IRIS) is inexpensive, simple to use, and amenable to microarray formats.

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, ...

Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers

The generation and detection of mechanical forces is a ubiquitous aspect of cell physiology, with direct relevance to cancer metastasis1, atherogenesis2 and wound healing3. In each of these examples, cells both exert force on their surroundings and simultaneously enzymatically remodel the extracellular matrix (ECM). The effect of forces on ECM has thus become an area of considerable interest due to its likely biological and medical importance4-7.
	Single molecule techniques such as optical trapping8, atomic force microscopy9, and magnetic tweezers10,11 allow researchers to probe the function of enzymes at a molecular level by exerting forces on individual proteins. Of these techniques, magnetic tweezers (MT) are notable for their low cost and high throughput. MT exert forces in the range of ~1-100 pN and can provide millisecond temporal resolution, qualities that are well matched to the study of enzyme mechanism at the single-molecule level12. Here we report a highly parallelizable MT assay to study the effect of force on the proteolysis of single protein molecules. We present the specific example of the proteolysis of a trimeric collagen peptide by matrix metalloproteinase 1 (MMP-1); however, this assay can be easily adapted to study other substrates and proteases.

In this article we describe the use of magnetic tweezers to study the effect of force on enzymatic proteolysis at the single molecule level in a highly parallelizable manner.

The  generation and detection of mechanical forces is a ubiquitous aspect of cell physiology,  with direct relevance to cancer metastasis1, atherogenesis2 ...

Bacterial Detection &amp; Identification Using Electrochemical Sensors

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

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

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

Sensing of Barrier Tissue Disruption with an Organic Electrochemical Transistor

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the passage of necessary ions and molecules. A breach in this barrier can be caused by a reduction in the extracellular calcium concentration. This reduction in calcium concentration causes a conformational change in proteins involved in the sealing of the barrier, leading to an increase of the paracellular flux. To mimic this effect the calcium chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N&#39;,N&#39;-tetra acetic acid (EGTA) was used on a monolayer of cells known to be representative of the gastrointestinal tract. Different methods to detect the disruption of the barrier tissue already exist, such as immunofluorescence and&#160;permeability assays. However, these methods are time-consuming and costly and not suited to dynamic or high-throughput measurements. Electronic methods for measuring barrier tissue integrity also exist for measurement of the transepithelial resistance (TER), however these are often costly and complex. The development of rapid, cheap, and sensitive methods is urgently needed as the integrity of barrier tissue is a key parameter in drug discovery and pathogen/toxin diagnostics. The organic electrochemical transistor (OECT) integrated with barrier tissue forming cells has been shown as a new device capable of dynamically monitoring barrier tissue integrity. The device is able to measure minute variations in ionic flux with unprecedented temporal resolution and sensitivity, in real time, as an indicator of barrier tissue integrity. This new method is based on a simple device that can be compatible with high throughput screening applications and fabricated at low cost.

The Organic Electrochemical Transistor is integrated with live cells and used to monitor ion flux across the gastrointestinal epithelial barrier. In this study, an increase in ion flux, related to disruption of tight junctions, induced by the presence of the calcium chelator EGTA (ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetra acetic acid), is measured.

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the ...

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 ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage dependent cells on the 2-dimensional surface of tissue culture plastic. More recently, there is a growing body of data demonstrating more in vivo-like behaviors of cells grown in 3-dimensional culture systems. This manuscript describes in detail the set-up and operation of a hollow fiber bioreactor system for the in vivo-like culture of mammalian cells. The hollow fiber bioreactor system delivers media to the cells in a manner akin to the delivery of blood through the capillary networks in vivo. The system is designed to fit onto the shelf of a standard CO2 incubator and is simple enough to be set-up by any competent cell biologist with a good understanding of aseptic technique. The systems utility is demonstrated by culturing the hepatocarcinoma cell line HepG2/C3A for 7 days. Further to this and in line with other published reports on the functionality of cells grown in 3-dimensional culture systems the cells are shown to possess increased albumin production (an important hepatic function) when compared to standard 2-dimensional tissue culture.

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

The functional behavior of cells in culture can be improved by culturing in more in vivo-like 3-dimensional culture environments16-21. This manuscript describes the set-up and operation of a hollow fiber bioreactor system for in vivo-like mammalian tissue culture.

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage ...

Extraction of Plant-based Capsules for Microencapsulation Applications

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin exine capsules (SECs) are obtained when spores or pollen are processed so as to remove the internal sporoplasmic contents. The resulting hollow microcapsules exhibit a high degree of micromeritic uniformity and retain intricate microstructural features related to the particular plant species. Herein, we demonstrate a streamlined process for the production of SECs from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs. The current SEC isolation procedure has been recently optimized to significantly reduce the processing requirements which are conventionally used in SEC isolation, and to ensure the production of intact microcapsules. Natural L. clavatum spores are defatted with acetone, treated with phosphoric acid, and extensively washed to remove sporoplasmic contents. After acetone defatting, a single processing step using 85% phosphoric acid has been shown to remove all sporoplasmic contents. By limiting the acid processing time to 30 hr, it is possible to isolate clean SECs and avoid SEC fracturing, which has been shown to occur with prolonged processing time. Extensive washing with water, dilute acids, dilute bases, and solvents ensures that all sporoplasmic material and chemical residues are adequately removed. The vacuum loading technique is utilized to load a model protein (Bovine Serum Albumin) as a representative hydrophilic compound. Vacuum loading provides a simple technique to load various compounds without the need for harsh solvents or undesirable chemicals which are often required in other microencapsulation protocols. Based on these isolation and loading protocols, SECs provide a promising material for use in a diverse range of microencapsulation applications, such as, therapeutics, foods, cosmetics, and personal care products.

This protocol/manuscript describes a streamlined process for the production of sporopollenin exine capsules (SECs) from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs.

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin ...

Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands of micro-scale immunoassays performed simultaneously on a single chip. Sandwich assay format improves assay specificity by detecting each target with two antibodies, but suffers from cross-reactivity between reagents thus limiting their multiplexing capabilities. Antibody colocalization microarray (ACM) has been developed for cross-reactivity-free multiplexed protein detection, but requires an expensive spotter on-site for microarray fabrication during assays. In this work, we demonstrate a snap chip technology that transfers reagent from microarray-to-microarray by simply snapping two chips together, thus no spotter is needed during the sample incubation and subsequent application of detection antibodies (dAbs) upon storage of pre-spotted slides, dissociating the slide preparation from assay execution. Both single and double transfer methods are presented to achieve accurate alignment between the two microarrays and the slide fabrication for both methods are described. Results show that &#60;40 &#956;m alignment has been achieved with double transfer, reaching an array density of 625 spots/cm2. A 50-plexed immunoassay has been conducted to demonstrate the usability of the snap chip in multiplexed protein analysis. Limits of detection of 35 proteins are in the range of pg/mL.

We demonstrate a snap chip technology for performing cross-reactivity-free multiplexed sandwich immunoassays by simply snapping two slides. A snap apparatus is used for reliably transferring reagents from microarray-to-microarray. The snap chip can be used for any biochemical reactions requiring colocalization of different reagents without cross-contamination.

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands ...

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, ...