Macrocycle peptides delineate locked-open inhibition mechanism for microorganism phosphoglycerate mutases.

Glycolytic interconversion of phosphoglycerate isomers is catalysed in numerous pathogenic microorganisms by a cofactor-independent mutase (iPGM) structurally distinct from the mammalian cofactor-dependent (dPGM) isozyme. The iPGM active site dynamically assembles through substrate-triggered movement of phosphatase and transferase domains creating a solvent inaccessible cavity. Here we identify alternate ligand binding regions using nematode iPGM to select and enrich lariat-like ligands from an mRNA-display macrocyclic peptide library containing >1012 members. Functional analysis of the ligands, named ipglycermides, demonstrates sub-nanomolar inhibition of iPGM with complete selectivity over dPGM. The crystal structure of an iPGM macrocyclic peptide complex illuminated an allosteric, locked-open inhibition mechanism placing the cyclic peptide at the bi-domain interface. This binding mode aligns the pendant lariat cysteine thiolate for coordination with the iPGM transition metal ion cluster. The extended charged, hydrophilic binding surface interaction rationalizes the persistent challenges these enzymes have presented to small-molecule screening efforts highlighting the important roles of macrocyclic peptides in expanding chemical diversity for ligand discovery.

Measurements of translation initiation from all 64 codons in E. coli.

Our understanding of translation underpins our capacity to engineer living systems. The canonical start codon (AUG) and a few near-cognates (GUG, UUG) are considered as the 'start codons' for translation initiation in Escherichia coli. Translation is typically not thought to initiate from the 61 remaining codons. Here, we quantified translation initiation of green fluorescent protein and nanoluciferase in E. coli from all 64 triplet codons and across a range of DNA copy number. We detected initiation of protein synthesis above measurement background for 47 codons. Translation from non-canonical start codons ranged from 0.007 to 3% relative to translation from AUG. Translation from 17 non-AUG codons exceeded the highest reported rates of non-cognate codon recognition. Translation initiation from non-canonical start codons may contribute to the synthesis of peptides in both natural and synthetic biological systems.

When Wavelengths Collide: Bias in Cell Abundance Measurements Due to Expressed Fluorescent Proteins.

The abundance of bacteria in liquid culture is commonly inferred by measuring optical density at 600 nm. Red fluorescent proteins (RFPs) can strongly absorb light at 600 nm. Increasing RFP expression can falsely inflate apparent cell density and lead to underestimations of mean per-cell fluorescence by up to 10%. Measuring optical density at 700 nm would allow estimation of cell abundance unaffected by the presence of nearly all fluorescent proteins.

Total protein quantitation using the bicinchoninic acid assay and gradient elution moving boundary electrophoresis.

We investigated the ability of gradient elution moving boundary electrophoresis (GEMBE) with capacitively coupled contactless conductivity detection (C(4) D) to assay total protein concentration using the bicinchoninic acid (BCA) reaction. We chose this format because GEMBE-C(4) D behaves as a concentration dependent detection system, unlike optical methods that also rely on pathlength (due to Beer's law). This system tolerates proteins well compared with other capillary electrophoretic methods, allowing the capillary to be reused without coatings or additional hydroxide wash steps. The typical reaction protocol was modified by reducing the pH slightly from 11.25 to 9.4, which enabled elimination of tartrate from the reagents. We estimated that copper (I) could be detected at approximately 3.0 µmol/L, which agrees with similar GEMBE and CZE systems utilizing C(4) D. Under conditions similar to the BCA "micro method" assay, we determined the LOD for three common proteins (insulin, BSA, and bovine gamma globulin) and found that they agree well with the existing spectroscopic detection methods. Further, we investigated how long reaction times impact the LOD and found that the conversion was proportional to log(time). This indicated that little sensitivity is gained by extending the reaction past 1 h. Hence, GEMBE provides an alternative platform for total protein assays while maintaining the excellent sensitivity of the optical-based methods.

Gradient elution moving boundary electrophoresis with channel current detection in relatively long capillaries.

A variant of gradient elution moving boundary electrophoresis with channel current detection is described, which uses relatively long channels (1.5 cm) for separation and detection. The signal for each analyte is determined to have the shape of a parabola ending with a break in slope. The capabilities of the new method are demonstrated for separation and quantitation of ATP and ADP, a measurement relevant for high-throughput screening assays. The results indicate that the new method is capable of reproducibly measuring the ratio of ADP concentration to ATP plus ADP concentration with an analysis time of less than 2 min and with a 1 SD uncertainty of 0.018 (over a range of 0-1).

Theoretical analysis of a magnetophoresis-diffusion T-sensor immunoassay.

We present the analytical investigation of a microfluidic homogeneous competitive immunoassay that incorporates antibody-conjugated superparamagnetic nanoparticles and magnetophoretic transport to enhance the limits of detection and dynamic range. The analytical model considers the advective, diffusive, and magnetophoretic transport of the antibody-coated nanoparticles relative to the labeled and sample antigens of interest in a T-sensor configuration. The magnetophoresis-diffusion immunoassay identified clear improvements to the assay response and reductions to the limit of detection for increased magnetophoretic velocities and larger nanoparticles. The externally applied magnetophoretic transport enriched the antibody-antigen accumulation region, while larger nanoparticles led to decreased diffusive peak broadening. The integration of nanoparticles to the diffusion immunoassay (NP-DIA) demonstrated an approximately 3-fold improvement to the limit of detection of the basic antibody/antigen system, while the integration of superparamagnetic nanoparticles and magnetophoretic transport (MIA) established an order of magnitude improvement in sensitivity as well as means to greatly reduce response time. The implementation of an external magnetic force enabled the detectable antigen size spectrum to extend from small molecules i.e., 10's Da to 100's Da, up to large proteins and macromolecules, i.e., 50 kDa to 150 kDa, for a single class of binding species, i.e., superparamagnetic nanoparticle. This investigation provides guidelines for the design and development of a magnetophoresis-diffusion T-sensor immunoassay, and clearly identifies the regimes for optimal operation.

Capturing rare cells from blood using a packed bed of custom-synthesized chitosan microparticles.

We describe batch generation of uniform multifunctional chitosan microparticles for isolation of rare cells, such as circulating tumor cells (CTCs), from a sample of whole blood. The chitosan microparticles were produced in large numbers using a simple and inexpensive microtubing arrangement. The particles were functionalized through encapsulation of carbon black, to control autofluorescence, and surface attachment of streptavidin, to enable interactions with biotinylated antibodies. These large custom modified microparticles (≈164 µm diameter) were then packed into a microfluidic channel to demonstrate their utility in rare cell capture. Blood spiked with breast cancer (MCF-7) cells was first treated with a biotinylated antibody (anti-EpCAM, which is selective for cancer cells like MCF-7) and then pumped through the device. In the process, the cancer cells were selectively bound to the microparticles through non-covalent streptavidin-biotin interactions. The number density of captured cells was determined by fluorescence microscopy at physiologically relevant levels. Selective capture of the MCF-7 cells was characterized, and compared favorably with previous approaches. The overall approach using custom synthesized microparticles is versatile, and can allow researchers more flexibility for rare cell capture through simpler and cheaper methods than are currently employed.

A simple packed bed device for antibody labelled rare cell capture from whole blood.

We have developed a system to isolate rare cells from whole blood using commercially available components and simple microfluidics. We characterized the capture of MCF-7 cells spiked into whole human blood using this system to demonstrate that enrichment and enumeration studies give results similar to in situ surface-modified devices while reducing fabrication and operation complexity.

Image-based feedback control for real-time sorting of microspheres in a microfluidic device.

We describe a control system to automatically distribute antibody-functionalized beads to addressable assay chambers within a PDMS microfluidic device. The system used real-time image acquisition and processing to manage the valve states required to sort beads with unit precision. The image processing component of the control system correctly counted the number of beads in 99.81% of images (2689 of 2694), with only four instances of an incorrect number of beads being sorted to an assay chamber, and one instance of inaccurately counted beads being improperly delivered to waste. Post-experimental refinement of the counting script resulted in one counting error in 2694 images of beads (99.96% accuracy). We analyzed a range of operational variables (flow pressure, bead concentration, etc.) using a statistical model to characterize those that yielded optimal sorting speed and efficiency. The integrated device was able to capture, count, and deliver beads at a rate of approximately four per minute so that bead arrays could be assembled in 32 individually addressable assay chambers for eight analytical measurements in duplicate (512 beads total) within 2.5 hours. This functionality demonstrates the successful integration of a robust control system with precision bead handling that is the enabling technology for future development of a highly multiplexed bead-based analytical device.

T7-based linear amplification of low concentration mRNA samples using beads and microfluidics for global gene expression measurements.

We have demonstrated in vitro transcription (IVT) of cDNA sequences from purified Jurkat T-cell mRNA immobilized on microfluidic packed beds down to single-cell quantities. The microfluidically amplified antisense-RNA (aRNA) was nearly identical in length and quantity compared with benchtop reactions using the same starting sample quantities. Microarrays were used to characterize the number and population of genes in each sample, allowing comparison of the microfluidic and benchtop processes. For both benchtop and microfluidic assays, we measured the expression of approximately 4000 to 9000 genes for sample amounts ranging from 20 pg to 10 ng (2 to 1000 cell equivalents), corresponding to 41 to 93% of the absolute number of genes detected from a 100 ng total RNA control sample. Concordance of genes detected between methods (benchtop vs. microfluidic) and repeats (microfluidic vs. microfluidic) typically exceeded 90%. Validation of microarray by Real-time PCR of a panel of five genes suggests transcription of genes present is approximately six times more efficient with the microfluidic IVT compared with benchtop processing. Microfluidic IVT introduces no bias to the gene expression profile of the sample and provides more efficient transcription of mRNA sequences present at the single-cell level.

Development of aptamer-based affinity assays using temperature gradient focusing: minimization of the limit of detection.

A method is described for an aptamer-based affinity assay using a combination of two nonconventional techniques, temperature gradient focusing (TGF) and field-amplified continuous sample injection TGF (FACSI-TGF), with fluorescence detection. Human immunodeficiency virus reverse transcriptase (HIVRT) is used as the protein target for the assay. The TGF and FACSI-TGF assays are compared to similar results obtained with conventional CE. A range of starting aptamer concentrations are used to determine the optimal LOD for human immunodeficiency virus reverse transcriptase (HIVRT) using each approach. The results indicate that the LODs for HIVRT obtained with TGF and FACSI-TGF are comparable to or even lower than the LODs obtained with conventional CE in spite of the inferior detector used for the TGF and FACSI-TGF assays (arc lamp and low-cost CCD for TGF versus LIF with PMT for CE). It is hypothesized that this is due to the greater reproducibility of the TGF and FACSI-TGF techniques since they do not employ a defined sample injection. The lowest LOD achieved with the new aptamer assay approach is more than an order of magnitude lower than that reported for a similar CE-based aptamer assay for the same target.

Counterflow rejection of adsorbing proteins for characterization of biomolecular interactions by temperature gradient focusing.

A new technique is described for the analysis of small molecules in samples containing serum proteins and for the measurement of the binding of small molecules to serum proteins. The new technique is based on temperature gradient focusing (TGF) and takes advantage of the counterflow used with TGF to exclude serum proteins from the analysis channel while small molecules are focused for detection. The technique is demonstrated for the measurement of the binding constant between a small molecule and serum albumin using both a direct measurement of the free fraction of the small molecule as well as using a competitive binding assay.

Temperature gradient focusing with field-amplified continuous sample injection for dual-stage analyte enrichment and separation.

We describe the serial combination of temperature gradient focusing (TGF) and field-amplified continuous sample injection (FACSI) for improved analyte enrichment and electrophoretic separation. TGF is a counterflow equilibrium gradient method for the simultaneous concentration and separation of analytes. When TGF is implemented with a low conductivity sample buffer and a (relatively) high conductivity separation buffer, a form of sample enrichment similar to field-amplified sample stacking (FASS) or field-amplified sample injection (FASI) is achieved in addition to the normal TGF sample enrichment. FACSI-TGF differs from FASI in two important respects: continuous sample injection, versus a discrete injection, is utilized; because of the counterflow employed for TGF, the stacking interface exists in a pseudo-stationary region outside of the separation column. Notably, analyte concentration enrichment factors greater than the ratio of separation and sample conductivities (gamma) were achieved in this method. For gamma=6.1, the concentration factor for one model analyte (Oregon Green 488) was found to be 36-fold higher with FACSI-TGF as compared to TGF without FACSI. A separation of five fluorescently labeled amino acids is also demonstrated with the technique, yielding an average enrichment of greater than 1000-fold.

Gradient elution moving boundary electrophoresis for high-throughput multiplexed microfluidic devices.

A novel method for performing electrophoretic separations is described-gradient elution moving boundary electrophoresis (GEMBE). The technique utilizes the electrophoretic migration of chemical species in combination with variable hydrodynamic bulk counterflow of the solution through a separation capillary or microfluidic channel. Continuous sample introduction is used, eliminating the need for a sample injection mechanism. Only analytes with an electrophoretic velocity greater than the counterflow velocity enter the separation channel. The counterflow velocity is varied over time so that each analyte is brought into the separation column at different times, allowing for high-resolution separations in very short channels. The new variable of bulk flow acceleration affords a new selectivity parameter to electrophoresis analogous to gradient elution compositions in chromatography. Because it does not require extra channels or access ports to form an injection zone and because separations can be performed in very short channels, GEMBE separations can be implemented in much smaller areas on a micro-fluidic chip as compared to conventional capillary electrophoresis. Demonstrations of GEMBE separations of small dye molecules, amino acids, DNA, and immunoassay products are presented. A low-cost, polymeric, eight-channel multiplexed microfluidic device was fabricated to demonstrate the reduced area requirements of GEMBE; the device was less than 1 in.2 in area and required only n + 1 fluidic access ports per n analyses (in this instance, nine ports for eight analyses). Parallel separations of fluorescein and carboxyfluorescein yielded less than 3% relative standard deviation (RSD) in interchannel migration times and less than 5% RSD in both peak and height measurements. The device was also used to generate a calibration curve for a homogeneous insulin immunoassay using each of the eight channels as a calibration point with less than 5% RSD at each point with replicate analyses.

Scanning temperature gradient focusing.

Temperature gradient focusing (TGF) is a recently developed technique for the simultaneous concentration and electrophoretic separation of ionic analytes in microfluidic channels. One drawback to TGF as it has previously been described is the limited peak capacity; only a small number of analyte peaks (approximately 2-3) can be simultaneously focused and separated. In this paper, we report on a variation of the TGF method whereby the bulk flow rate is varied over time so that a large number of analytes can be sequentially focused, moved past a fixed detection point, and flushed to waste. In addition to improved peak capacity, the detection limits of the scanning TGF method can be adjusted on-the-fly, as needed for different samples. Finally, scanning TGF provides a technique by which high-resolution, high-peak-capacity electrophoretic separations can be performed in simple, straight, and short microfluidic channels.

Quantitative temperature gradient focusing performed using background electrolytes at various pH values.

Temperature gradient focusing (TGF) has previously been shown to be a practical technique for simultaneous concentration and separation of a variety of samples. In this paper, we demonstrate that TGF can be conducted at a wide range of pH values. Techniques for first-order prediction of the suitability of a given BGE for focusing are discussed. Buffer suitability for TGF is assessed experimentally by simultaneously concentrating and separating a pair of fluorescent analytes. One analyte is held at constant concentration for use as an internal standard while the concentration of the other dye is varied. Peak area is shown to vary linearly with the input dye concentration. A high degree of resolution (R(s) >3) of fluorescein and carboxyfluorescein, as well as for two LysoSensor-based dyes, is also observed. Foucusing and separation by TGF was successfully conducted quantitatively in BGE solutions of pH from 3.0 to 10.5.

On-chip titration of an anticoagulant argatroban and determination of the clotting time within whole blood or plasma using a plug-based microfluidic system.

This paper describes extending plug-based microfluidics to handling complex biological fluids such as blood, solving the problem of injecting additional reagents into plugs, and applying this system to measuring of clotting time in small volumes of whole blood and plasma. Plugs are droplets transported through microchannels by fluorocarbon fluids. A plug-based microfluidic system was developed to titrate an anticoagulant (argatroban) into blood samples and to measure the clotting time using the activated partial thromboplastin time (APTT) test. To carry out these experiments, the following techniques were developed for a plug-based system: (i) using Teflon AF coating on the microchannel wall to enable formation of plugs containing blood and transport of the solid fibrin clots within plugs, (ii) using a hydrophilic glass capillary to enable reliable merging of a reagent from an aqueous stream into plugs, (iii) using bright-field microscopy to detect the formation of a fibrin clot within plugs and using fluorescent microscopy to detect the production of thrombin using a fluorogenic substrate, and (iv) titration of argatroban (0-1.5 microg/mL) into plugs and measurement of the resulting APTTs at room temperature (23 degrees C) and physiological temperature (37 degrees C). APTT measurements were conducted with normal pooled plasma (platelet-poor plasma) and with donor's blood samples (both whole blood and platelet-rich plasma). APTT values and APTT ratios measured by the plug-based microfluidic device were compared to the results from a clinical laboratory at 37 degrees C. APTT obtained from the on-chip assay were about double those from the clinical laboratory but the APTT ratios from these two methods agreed well with each other.

Characterization of the local temperature in space and time around a developing Drosophila embryo in a microfluidic device.

This paper characterizes a microfluidic platform that differentially controls the temperature of each half of a living Drosophila melanogaster fruitfly embryo in space and time (E. M. Lucchetta, J. H. Lee, L. A. Fu, N. H. Patel and R. F. Ismagilov, Nature, 2005, 434, 1134-1138). This platform relies on laminar flow of two streams of liquid with different temperature, and on rapid prototyping in polydimethylsiloxane (PDMS). Here, we characterized fluid flow and heat transport in this platform both experimentally and by numerical simulation, and estimated the temperature distribution around and within the embryo by numerical simulation, to identify the conditions for creating a sharper temperature difference (temperature step) over the embryo. Embryos were removed from the device and immunostained histochemically for detection of Paired protein. Biochemical processes are sensitive to small differences in environmental temperature. The microfluidic platform characterized here could prove useful in understanding dynamics of biochemical networks as they respond to changes in temperature.

Diffusion based analysis in a sheath flow microchannel: the sheath flow T-sensor.

This paper describes a microfluidic channel that allows for diffusion-based analysis of adsorbing species without passivation of the channel surfaces. The sheath flow configuration was used to measure the diffusion coefficient of fluorescently labeled species from their spatial distribution within the microchannel by analyzing the derivative of the intensity profile at the interface between two distinct core fluids. Measurements for both a small molecule (rhodamine B) and an intermediate-sized protein (wheat germ agglutinin) were made, demonstrating the utility of the sheath flow T-sensor.