A sensor capable of continuously measuring specific molecules in the bloodstream in vivo would give clinicians a valuable window into patients’ health and their response to therapeutics. Such technology would enable truly personalized medicine, wherein therapeutic agents could be tailored with optimal doses for each patient to maximize efficacy and minimize side effects. Unfortunately, continuous, real-time measurement is currently only possible for a handful of targets, such as glucose, lactose, and oxygen, and the few existing platforms for continuous measurement are not generalizable for the monitoring of other analytes, such as small-molecule therapeutics. In response, we have developed a real-time biosensor capable of continuously tracking a wide range of circulating drugs in living subjects. Our microfluidic electrochemical detector for in vivo continuous monitoring (MEDIC) requires no exogenous reagents, operates at room temperature, and can be reconfigured to measure different target molecules by exchanging probes in a modular manner. To demonstrate the system's versatility, we measured therapeutic in vivo concentrations of doxorubicin (a chemotherapeutic) and kanamycin (an antibiotic) in live rats and in human whole blood for several hours with high sensitivity and specificity at sub-minute temporal resolution. Importantly, we show that MEDIC can also obtain pharmacokineticparameters for individual animals in real-time. Accordingly, just as continuous glucose monitoring technology is currently revolutionizing diabetes care, we believe MEDIC could be a powerful enabler for personalized medicine by ensuring delivery of optimal drug doses for individual patients based on direct detection of physiological parameters.

Although potentiostats are the foundation of modern electrochemical research, they have seen relatively little application in resource poor settings, such as undergraduate laboratory courses and the developing world. One reason for the low penetration of potentiostats is their cost, as even the least expensive commercially available laboratory potentiostats sell for more than one thousand dollars. An inexpensive electrochemical workstation could thus prove useful in educational labs, and increase access to electrochemistry-based analytical techniques for food, drug and environmental monitoring. With these motivations in mind, we describe here the CheapStat, an inexpensive (<$80), open-source (software and hardware), hand-held potentiostat that can be constructed by anyone who is proficient at assembling circuits. This device supports a number of potential waveforms necessary to perform cyclic, square wave, linear sweep and anodic stripping voltammetry. As we demonstrate, it is suitable for a wide range of applications ranging from food- and drug-quality testing to environmental monitoring, rapid DNA detection, and educational exercises. The device's schematics, parts lists, circuit board layout files, sample experiments, and detailed assembly instructions are available in the supporting information and are released under an open hardware license.

Practical and high-throughput assays for probing protein-ligand interactions are essential for proteomics and drug development. 1 For example, the analysis of multiprotein complexes involved in gene regulation is a combinatorial challenge with applications in medical diagnostics. 2 Here we describe an approach using surfaceenhanced resonance Raman scattering (SERRS) for protein sensing in a tightly controlled assembly of gold nanoparticles and DNA, which has great potential for high sensitivity with high-throughput multiplexing capacity. 3 SERRS techniques greatly enhance signal strength and sensitivity in many applications, with demonstrations of detection limits at the single-molecule level, 4,5 while offering other important benefits over fluorescent detection methods, including resistance to photobleaching and narrow emission peaks for spectral multiplexing. 6 However, the enhancement possible from SERRS is very dependent on the distance between, the surface morphology of, and the optical resonance of closely associated metal nanoparticles, making the design of controlled assemblies paramount to correctly position analytes for optimal detection. 7 We describe an effective architecture of DNA-bridged nanoparticle assemblies for binding and detecting sequence and concentration dependent protein-DNA interactions. Each short stretch of duplex DNA, which is to be bound by the analyte protein, is prepared with overhangs that hybridize and cross-link a generic set of gold nanoparticles (NPs) functionalized with complementary DNA. 8 This self-assembling scaffold allows control of the positioning of metallic NPs to directly surround a DNA sequence recognized by an analyte protein (tagged with a resonance Raman molecule). These NPs are subsequently grown using a silver plating step to decrease the distance between the surfaces and the analyte causing a large increase in SERRS signal, detected by a confocal Raman microprobe. 5,9 The assembly consists of a three-part oligonucleotide scaffolding tethering NPs as shown in Figure 1A. Double-stranded oligonucleotides C (oligo-C) of lengths 15 to 39 base pairs containing the protein binding site of interest were designed to generate appropriate spacing for protein access into the final assemblies, with 12 base pair single-stranded overhangs on each end that are complementary to surface-bound 22 base pair oligonucleotides A or B (oligo-A or oligo-B, DNA sequences in Supporting Information). Gold NPs diameter of ∼13 nm were prepared by citrate reduction of gold aurate, 11 and the resultant citrate shell was displaced by thiolmodified oligo-A or oligo-B 10 . Conjugates were determined to have 183 ( 20 oligonucleotides per particle (Supporting Information). Nanoparticles of this size have previously been used as seeds for silver plating and multiplexed detection by SERRS. 11 Upon annealing of the single-stranded overhangs, the three components condense into assemblies. 10 Variations of assemblies were formed with oligo-C containing the GCGC recognition site of M.HhaI, 12 the T...

Human induced pluripotent stem cells (iPSCs) hold promise as a source of adult-derived, patient-specific pluripotent cells for use in cell-based regenerative therapies. However, current methods of cell culture are tedious and expensive, and the mechanisms underlying cell proliferation are not understood. In this study, we investigated expression and function of iPSC integrin extracellular matrix receptors to better understand the molecular mechanisms of cell adhesion, survival, and proliferation. We show that iPSC lines generated using Oct-3/4, Sox-2, Nanog, and Lin-28 express a repertoire of integrins similar to that of hESCs, with prominent expression of subunits alpha5, alpha6, alphav, beta1, and beta5. Integrin function was investigated in iPSCs cultured without feeder layers on Matrigel or vitronectin, in comparison to human embryonic stem cells. beta1 integrins were required for adhesion and proliferation on Matrigel, as shown by immunological blockade experiments. On vitronectin, the integrin alphavbeta5 was required for initial attachment, but inhibition of both alphavbeta5 and beta1 was required to significantly decrease iPSC proliferation. Furthermore, iPSCs cultured on vitronectin for 9 passages retained normal karyotype, pluripotency marker expression, and capacity to differentiate in vitro. These studies suggest that vitronectin, or derivatives thereof, might substitute for Matrigel in a more defined system for iPSC culture.

Transcription factor expression levels, which sensitively reflect cellular development and disease state, are typically monitored via cumbersome, reagent-intensive assays that require relatively large quantities of cells. Here we demonstrate a simple, quantitative approach to their detection based on a simple, electrochemical sensing platform. This sensor sensitively and quantitatively detects its target transcription factor in complex media (e.g., 250 μg/ml crude nuclear extracts) in a convenient, low-reagent process requiring only 10 μl of sample. Our approach thus appears a promising means of monitoring transcription factor levels.

Here we demonstrate a reagentless, electrochemical platform for the specific detection of proteins that bind to single-or double-stranded DNA. The sensor is composed of a double-or single-stranded, redox-tagged DNA probe which is covalently attached to an interrogating electrode. Upon protein binding the current arising from the redox tag is suppressed, indicating the presence of the target. Using this approach we have fabricated sensors against the double-stranded DNA binding proteins TATA-box binding protein and M.HhaI methyltransferase, and against the single-strand binding proteins Escherichia coli SSBP and replication protein A. All four targets are detected at nanomolar concentrations, in minutes, and in a convenient, general, readily reusable, electrochemical format. The approach is specific; we observed no significant cross-reactivity between the sensors. Likewise the approach is selective; it supports, for example, the detection of single strand binding protein directly in crude nuclear extracts. The generality of our approach (including its ability to detect both double-and single-strand binding proteins) and a strong, non-monotonic dependence of signal gain on probe density support a collisional signaling mechanism in which binding alters the collision efficiency, and thus electron transfer efficiency, of the attached redox tag. Given the ubiquity with which protein binding will alter the collisional dynamics of an oligonucleotide, we believe this approach may prove of general utility in the detection of DNA and RNA binding proteins.The reagentless, electrochemical E-DNA (electrochemical DNA) [reviewed in Ricci and Plaxco, 2008] 1 and E-AB (electrochemical, aptamer-based) [reviewed in Xiao et al., 2008] 2 sensing platforms are a promising approach for the detection of a wide range of molecular analytes. 3-5 Composed of an electrode-bound, redox-modified probe oligonucleotide, E-DNA and E-AB sensors require only that target binding alters the rates with which the probe-attached redox-tag collides with, and thus transfers electrons to, the interrogating electrode. 6,7 Because all of the sensing components in the E-DNA/E-AB platform are tightly linked to the electrode, these sensors are reagentless and readily reusable. 8 Likewise, because their signaling is linked to a binding-specific change in the properties of the probe DNA, and not simply to adsorption to the sensor surface, E-DNA and E-AB sensors have proven remarkably robust against the MATERIALS AND METHODS Probe DNA SequencesWe have employed the following probe DNA sequences:TATA probe: 5′-HS-(CH 2 ) 6 -CGGGCTATAT*(MB) AAGGGGCGTTTTCTTATATAG-3′ M.HhaI probe: 5′-HS-(CH 2 ) 6 -AAGACGAGCGCATGTT*(MB)-TATGCGCTC-3′Poly-T 20 probe: 5′-HS-(CH 2 ) 6 -T 20 -(CH 2 ) 7 -NH-MB-3′ Poly-T 40 probe: 5′-HS-(CH 2 ) 6 -T 40 -(CH 2 ) 7 -NH-MB-3′Poly-T 70 probe:where -(CH 2 ) 7 -NH-MB-3′ represents a methylene blue (MB) added to the terminal phosphate via a C-7 amino linker and T*(MB) represents a thymine nucleotide modified by the addition of MB to a 6-c...

The development of convenient, real-time probes for monitoring protein function in biological samples represents an important challenge of the postgenomic era. In response, we introduce here “transcription factor beacons,” binding-activated fluorescent DNA probes that signal the presence of specific DNA-binding activities. As a proof of principle, we present beacons for the rapid, sensitive detection of three transcription factors (TATA Binding Protein, Myc-Max, and NF-κB), and measure binding activity directly in crude nuclear extracts.

We have developed a high-throughput protein binding microarray (PBM) assay to systematically investigate transcription regulatory protein complexes binding to DNA with varied specificity and affinity. Our approach is based on the novel coupling of total internal reflectance fluorescence (TIRF) spectroscopy, swellable hydrogel double-stranded DNA microarrays and dye-labeled regulatory proteins, making it possible to determine both equilibrium binding specificities and kinetic rates for multiple protein:DNA interactions in a single experiment. DNA specificities and affinities for the general transcription factors TBP, TFIIA and IIB determined by TIRF–PBM are similar to those determined by traditional methods, while simultaneous measurement of the factors in binary and ternary protein complexes reveals preferred binding combinations. TIRF–PBM provides a novel and extendible platform for multi-protein transcription factor investigation.