Matthew S. Munson

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.

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

Abstract 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.

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.

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 (C4D) to assay total protein concentration using the bicinchoninic acid (BCA) reaction. We chose this format because GEMBE‐C4D behaves as a concentration dependent detection system, unlike optical methods that also rely on pathlength (due to Beers 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 C4D. 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.

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.

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.

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., 10s Da to 100s 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.

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).

Abstract 4070: Development of circulating tumor cell capture from whole blood using beads and microfluidics

Capture of circulating tumor cells (CTCs) from blood shows promise as a relatively non-invasive screen for early stage metastasis, treatment efficacy, and disease progression. Capture rates exceeding 80% of CTCs from whole blood have been demonstrated in microfluidic chips, with good specificity and throughput at approximately 1 mL/h. The most common approach is to design a high surface-area microdevice and functionalize the surface with anti-EpCAM to capture epithelial tumor cells. As an alternative to this monolithic design, we have developed a CTC capture strategy based on functionalized beads for analyzing small numbers of CTCs from whole blood. Beads for the device were commercially available in a variety of sizes and surface chemistries or could be synthesized by multiple techniques. We utilized avidin-functionalized beads to enable capture using biotinylated antibodies (Ab) for surface-expressed proteins. Critically, batches of beads were assayed in bulk and then used for multiple experiments, avoiding characterization of each device. Beads packed well as columns in microdevices, ensuring multiple surface interactions as cells flow through. We simulated CTC-carrying blood by spiking human breast cancer adenocarcinoma (MFC7) cells (prestained with a membrane dye) into whole human blood between 1000 cells/mL and 105 cells/mL. Samples were delivered through the packed bed of beads using a syringe pump. We found that adding Ab to whole blood samples provided superior results to pre-coating the beads with the Ab. This approach was tested for multiple bead compositions and enabled (1) use of less Ab, and (2) higher capture rates, perhaps due to the rapid biotin:avidin binding. Significant cell capture was observed when as few as 300 cells were introduced into the device. At all cell densities, the majority of captured cells bound to the first few rows of beads, with decreasing capture frequency along the length of the column. Quantification of the absolute number of captured cells was a challenge in whole blood samples due to the large numbers of cells captured and optical distortion effects. We estimated capture by observing the initial capture rates of the same cells at the same densities in serum-spiked phosphate-buffered saline. This indicated that during the initial stages of flow, over 80% of the Ab-labeled cells were selectively captured. We conclude that the device can handle higher flowrates and still capture the majority of labeled cells. We are exploring the feasibility of this approach for capturing even lower CTC concentrations (1 cell/ml to 100 cells/mL range). Our bead/microfluidic approach simplifies CTC capture by enabling bulk surface functionalization for multiple experiments and allowing commercial sourcing of all functional elements. Further, there is flexibility in antibody selection, such that alternate Ab could rapidly be considered. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4070. doi:1538-7445.AM2012-4070