Gregory L. Holst

Time-Elapse Communication: Bacterial Communication on a Microfluidic Chip

Bacterial populations housed in microfluidic environments can serve as transceivers for molecular communication, but the data-rates are extremely low (e.g., 10-5 bits per second.). In this work, genetically engineered Escherichia coli bacteria were maintained in a microfluidic device where their response to a chemical stimulus was examined over time. The bacteria serve as a communication receiver where a simple modulation such as on-off keying (OOK) is achievable, although it suffers from very poor data-rates. We explore an alternative communication strategy called time-elapse communication (TEC) that uses the time period between signals to encode information. We identify the limitations of TEC under practical non-zero error conditions and propose an advanced communication strategy called smart time-elapse communication (TEC-SMART) that achieves over a 10x improvement in data-rate over OOK. We derive the capacity of TEC and provide a theoretical maximum data-rate that can be achieved.

Rapid, quantitative, reverse transcription PCR in a polymer microfluidicchip

Quantitative PCR (qPCR) techniques have become invaluable, high-throughput tools to study gene expression. However, the need to measure gene expression patterns quickly and affordably, useful for applications such as stem cell biomanufacturing requiring real-time observation and control, has not been adequately met by rapid qPCR instrumentation to date. We report a reverse transcription, microfluidic qPCR system and its application to DNA and RNA amplification measurement. In the system, an environmental control fixture provides mechanical and thermal repeatability for an infrared laser to achieve both accurate and precise open-loop temperature control of 1 μl reaction volumes in a low-cost polymer microfluidic chip with concurrent fluorescence imaging. We have used this system to amplify serial dilutions of λ-phage DNA (10(5)-10(7) starting copies) and RNA transcripts from the GAPDH housekeeping gene (5.45 ng total mouse embryonic stem cell RNA) and measured associated standard curves, efficiency (57%), repeatability (~1 cycle threshold), melting curves, and specificity. This microfluidic qRT-PCR system offers a practical approach to rapid analysis (~1 h), combining the cost benefits of small reagent volumes with the simplicity of disposable polymer microchips and easy setup.

Thermally multiplexed polymerase chain reaction.

Amplification of multiple unique genetic targets using the polymerase chain reaction (PCR) is commonly required in molecular biology laboratories. Such reactions are typically performed either serially or by multiplex PCR. Serial reactions are time consuming, and multiplex PCR, while powerful and widely used, can be prone to amplification bias, PCR drift, and primer-primer interactions. We present a new thermocycling method, termed thermal multiplexing, in which a single heat source is uniformly distributed and selectively modulated for independent temperature control of an array of PCR reactions. Thermal multiplexing allows amplification of multiple targets simultaneously-each reaction segregated and performed at optimal conditions. We demonstrate the method using a microfluidic system consisting of an infrared laser thermocycler, a polymer microchip featuring 1 μl, oil-encapsulated reactions, and closed-loop pulse-width modulation control. Heat transfer modeling is used to characterize thermal performance limitations of the system. We validate the model and perform two reactions simultaneously with widely varying annealing temperatures (48 °C and 68 °C), demonstrating excellent amplification. In addition, to demonstrate microfluidic infrared PCR using clinical specimens, we successfully amplified and detected both influenza A and B from human nasopharyngeal swabs. Thermal multiplexing is scalable and applicable to challenges such as pathogen detection where patients presenting non-specific symptoms need to be efficiently screened across a viral or bacterial panel.

When bacteria talk: Time elapse communication for super-slow networks

In this work we consider nano-scale communication using bacterial populations as transceivers. We demonstrate using a microfluidic test-bed and a population of genetically engineered Escherichia coli bacteria serving as the communication receiver that a simple modulation like on-off keying (OOK) is indeed achievable, but suffers from very poor data-rates. We explore an alternative communication strategy called time elapse communication (TEC) that uses the time period between signals to encode information. We identify the severe limitations of TEC under practical non-zero error conditions in the target environment, and propose an advanced communication strategy called smart time elapse communication (TEC-SMART) that achieves over a 10× improvement in data-rate over OOK.

Robotic navigation to subcortical neural tissue for intracellular electrophysiology in vivo

In vivo studies of neurophysiology using the whole cell patch-clamp technique enable exquisite access to both intracellular dynamics and cytosol of cells in the living brain but are underrepresented in deep subcortical nuclei because of fouling of the sensitive electrode tip. We have developed an autonomous method to navigate electrodes around obstacles such as blood vessels after identifying them as a source of contamination during regional pipette localization (RPL) in vivo. In mice, robotic navigation prevented fouling of the electrode tip, increasing RPL success probability 3 mm below the pial surface to 82% (n = 72/88) over traditional, linear localization (25%, n = 24/95), and resulted in high-quality thalamic whole cell recordings with average access resistance (32.0 MΩ) and resting membrane potential (-62.9 mV) similar to cortical recordings in isoflurane-anesthetized mice. Whole cell yield improved from 1% (n = 1/95) to 10% (n = 9/88) when robotic navigation was used during RPL. This method opens the door to whole cell studies in deep subcortical nuclei, including multimodal cell typing and studies of long-range circuits.NEW & NOTEWORTHY This work represents an automated method for accessing subcortical neural tissue for intracellular electrophysiology in vivo. We have implemented a novel algorithm to detect obstructions during regional pipette localization and move around them while minimizing lateral displacement within brain tissue. This approach leverages computer control of pressure, manipulator position, and impedance measurements to create a closed-loop platform for pipette navigation in vivo. This technique enables whole cell patching studies to be performed throughout the living brain.