Non-invasive and accurate access of biomarkers remains a holy grail of the biomedical community. Human eccrine sweat is a surprisingly biomarker-rich fluid which is gaining increasing attention. This is especially true in applications of continuous bio-monitoring where other biofluids prove more challenging, if not impossible. However, much confusion on the topic exists as the microfluidics of the eccrine sweat gland has never been comprehensively presented and models of biomarker partitioning into sweat are either underdeveloped and/or highly scattered across literature. Reported here are microfluidic models for eccrine sweat generation and flow which are coupled with review of blood-to-sweat biomarker partition pathways, therefore providing insights such as how biomarker concentration changes with sweat flow rate. Additionally, it is shown that both flow rate and biomarker diffusion determine the effective sampling rate of biomarkers at the skin surface (chronological resolution). The discussion covers a broad class of biomarkers including ions (Na(+), Cl(-), K(+), NH4 (+)), small molecules (ethanol, cortisol, urea, and lactate), and even peptides or small proteins (neuropeptides and cytokines). The models are not meant to be exhaustive for all biomarkers, yet collectively serve as a foundational guide for further development of sweat-based diagnostics and for those beginning exploration of new biomarker opportunities in sweat.

Wearable digital health devices are dominantly found in rigid form factors such as bracelets and pucks. An adhesive radio-frequency identification (RFID) sensor bandage (patch) is reported, which can be made completely intimate with human skin, a distinct advantage for chronological monitoring of biomarkers in sweat. In this demonstration, a commercial RFID chip is adapted with minimum components to allow potentiometric sensing of solutes in sweat, and surface temperature, as read by an Android smartphone app with 96% accuracy at 50 mM Na(+) (in vitro tests). All circuitry is solder-reflow integrated on a standard Cu/polyimide flexible-electronic layer including an antenna, but while also allowing electroplating for simple integration of exotic metals for sensing electrodes. Optional paper microfluidics wick sweat from a sweat porous adhesive allowing flow to the sensor, or the sensor can be directly contacted to the skin. The wearability of the patch has been demonstrated for up to seven days, and includes a protective textile which provides a feel and appearance similar to a standard Band-Aid. Applications include hydration monitoring, but the basic capability is extendable to other mM ionic solutes in sweat (Cl(-), K(+), Mg(2+), NH4(+), and Zn(2+)). The design and fabrication of the patch are provided in full detail, as the basic components could be useful in the design of other wearable sensors.

Recent advances in cell-free gene expression (CFE) systems have enabled their use for a host of synthetic biology applications, particularly for rapid prototyping of genetic circuits and biosensors. Despite the proliferation of cell-free protein synthesis platforms, the large number of currently existing protocols for making CFE extracts muddles the collective understanding of how the extract preparation method affects its functionality. A key aspect of extract performance relevant to many applications is the activity of the native host transcriptional machinery that can mediate protein synthesis. However, protein yields from genes transcribed in vitro by the native Escherichia coli RNA polymerase are variable for different extract preparation techniques, and specifically low in some conventional crude extracts originally optimized for expression by the bacteriophage transcriptional machinery. Here, we show that cell-free expression of genes under bacterial σ70 promoters is constrained by the rate of transcription in crude extracts, and that processing the extract with a ribosomal runoff reaction and subsequent dialysis alleviates this constraint. Surprisingly, these processing steps only enhance protein synthesis in genes under native regulation, indicating that the translation rate is unaffected. We further investigate the role of other common extract preparation process variants on extract performance and demonstrate that bacterial transcription is inhibited by including glucose in the growth culture but is unaffected by flash-freezing the cell pellet prior to lysis. Our final streamlined and detailed protocol for preparing extract by sonication generates extract that facilitates expression from a diverse set of sensing modalities including protein and RNA regulators. We anticipate that this work will clarify the methodology for generating CFE extracts that are active for biosensing using native transcriptional machinery and will encourage the further proliferation of cell-free gene expression technology for new applications.

Advances in biosensor engineering have enabled the design of programmable molecular systems to detect a range of pathogens, nucleic acids, and chemicals. Here, we engineer and field-test a biosensor for fluoride, a major groundwater contaminant of global concern. The sensor consists of a cell-free system containing a DNA template that encodes a fluoride-responsive riboswitch regulating genes that produce a fluorescent or colorimetric output. Individual reactions can be lyophilized for long-term storage and detect fluoride at levels above 2 ppm, the Environmental Protection Agency's most stringent regulatory standard, in both laboratory and field conditions. Through onsite detection of fluoride in a real-world water source, this work provides a critical proof-ofprinciple for the future engineering of riboswitches and other biosensors to address challenges for global health and the environment.

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