Jason G. Kralj

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.

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.

Characterization of in vitro transcription amplification linearity and variability in the low copy number regime using External RNA Control Consortium (ERCC) spike-ins

Using spike-in controls designed to mimic mammalian mRNA species, we used the quantitative reverse transcription polymerase chain reaction (RT-qPCR) to assess the performance of in vitro transcription (IVT) amplification process of small samples. We focused especially on the confidence of the transcript level measurement, which is essential for differential gene expression analyses. IVT reproduced gene expression profiles down to approximately 100 absolute input copies. However, a RT-qPCR analysis of the antisense RNA showed a systematic bias against low copy number transcripts, regardless of sequence. Experiments also showed that noise increases with decreasing copy number. First-round IVT preserved the gene expression information within a sample down to the 100 copy level, regardless of total input sample amount. However, the amplification was nonlinear under low total RNA input/long IVT conditions. Variability of the amplification increased predictably with decreasing input copy number. For the small enrichments of interest in typical differential gene expression studies (e.g., twofold changes), the bias from IVT reactions is unlikely to affect the results. In limited cases, some transcript-specific differential gene expression values will need adjustment to reflect this bias. Proper experimental design with reasonable detection limits will yield differential gene expression capability even between low copy number transcripts.

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