Drug screening on digital microfluidics for cancer precision medicine.

Drug screening based on in-vitro primary tumor cell culture has demonstrated potential in personalized cancer diagnosis. However, the limited number of tumor cells, especially from patients with early stage cancer, has hindered the widespread application of this technique. Hence, we developed a digital microfluidic system for drug screening using primary tumor cells and established a working protocol for precision medicine. Smart control logic was developed to increase the throughput of the system and decrease its footprint to parallelly screen three drugs on a 4 × 4 cm2 chip in a device measuring 23 × 16 × 3.5 cm3. We validated this method in an MDA-MB-231 breast cancer xenograft mouse model and liver cancer specimens from patients, demonstrating tumor suppression in mice/patients treated with drugs that were screened to be effective on individual primary tumor cells. Mice treated with drugs screened on-chip as ineffective exhibited similar results to those in the control groups. The effective drug identified through on-chip screening demonstrated consistency with the absence of mutations in their related genes determined via exome sequencing of individual tumors, further validating this protocol. Therefore, this technique and system may promote advances in precision medicine for cancer treatment and, eventually, for any disease.

Micro-Electro-Mechanical Systems Microphones: A Brief Review Emphasizing Recent Advances in Audible Spectrum Applications.

The MEMS microphone is a representative device among the MEMS family, which has attracted substantial research interest, and those tailored for human voice have earned distinct success in commercialization. Although sustained development persists, challenges such as residual stress, environmental noise, and structural innovation are posed. To collect and summarize the recent advances in this subject, this paper presents a concise review concerning the transduction mechanism, diverse mechanical structure topologies, and effective methods of noise reduction for high-performance MEMS microphones with a dynamic range akin to the audible spectrum, aiming to provide a comprehensive and adequate analysis of this scope.

Miniature Magnetic Resonance Imaging System for in Situ Monitoring of Bacterial Growth and Biofilm Formation.

In situ monitoring of bacterial growth can greatly benefit human healthcare, biomedical research, and hygiene management. Magnetic resonance imaging (MRI) offers two key advantages in tracking bacterial growth: non-invasive monitoring through opaque sample containers and no need for sample pretreatment such as labeling. However, the large size and high cost of conventional MRI systems are the roadblocks for in situ monitoring. Here, we proposed a small, portable MRI system by combining a small permanent magnet and an integrated radio-frequency (RF) electronic chip that excites and reads out nuclear spin motions in a sample, and utilize this small MRI platform for in situ imaging of bacterial growth and biofilm formation. We demonstrate that MRI images taken by the miniature--and thus broadly deployable for in situ work--MRI system provide information on the spatial distribution of bacterial density, and a sequential set of MRI images taken at different times inform the temporal change of the spatial map of bacterial density, showing bacterial growth.

pH Regulator on Digital Microfluidics with Pico-Dosing Technique.

Real-time pH control on-chip is a crucial factor for cell-based experiments in microfluidics, yet difficult to realize. In this paper, we present a flexible pH regulator on a digital microfluidic (DMF) platform. The pico-dosing technology, which can generate and transfer satellite droplets, is presented to deliver alkali/acid into the sample solution to change the pH value of the sample. An image analysis method based on ImageJ is developed to calculate the delivered volume and an on-chip colorimetric method is proposed to determine the pH value of the sample solution containing the acid-base indicator. The calculated pH values show consistency with the measured ones. Our approach makes the real-time pH control of the on-chip biological experiment more easy to control and flexible.

A syndromic diagnostic assay on a macrochannel-to-digital microfluidic platform for automatic identification of multiple respiratory pathogens.

The worldwide COVID-19 pandemic has changed people's lives and the diagnostic landscape. The nucleic acid amplification test (NAT) as the gold standard for SARS-CoV-2 detection has been applied in containing its transmission. However, there remains a lack of an affordable on-site detection system at resource-limited areas. In this study, a low cost "sample-in-answer-out" system incorporating nucleic acid extraction, purification, and amplification was developed on a single macrochannel-to-digital microfluidic chip. The macrochannel fluidic subsystem worked as a world-to-chip interface receiving 500-1000 µL raw samples, which then underwent bead-based extraction and purification processes before being delivered to DMF. Electrodes actuate an eluent dispensed to eight independent droplets for reverse transcription quantitative polymerase chain reaction (RT-qPCR). By reading with 4 florescence channels, the system can accommodate a maximum of 32 detection targets. To evaluate the proposed platform, a comprehensive assessment was conducted on the microfluidic chip as well as its functional components (i.e., extraction and amplification). The platform demonstrated a superior performance. In particular, using clinical specimens, the chip targeting SARS-CoV-2 and Flu A/B exhibited 100% agreement with off-chip diagnoses. Furthermore, the fabrication of chips is ready for scaled-up manufacturing and they are cost-effective for disposable use since they are assembled using a printed circuit board (PCB) and prefabricated blocks. Overall, the macrochannel-to-digital microfluidic platform coincides with the requirements of point-of-care testing (POCT) because of its advantages: low-cost, ease of use, comparable sensitivity and specificity, and availability for mass production.

Sub-5-Minute Ultrafast PCR using Digital Microfluidics.

The development of a rapid and reliable polymerase chain reaction (PCR) method for point-of-care (POC) diagnosis is crucial for the timely identification of pathogens. Microfluidics, which involves the manipulation of small volumes of fluidic samples, has been shown to be an ideal approach for POC analysis. Among the various microfluidic platforms available, digital microfluidics (DMF) offers high degree of configurability in manipulating µL/nL-scale liquid and achieving automation. However, the successful implementation of ultrafast PCR on DMF platforms presents challenges due to inherent system instability. In this study, we developed a robust and ultrafast PCR in 3.7-5 min with a detection sensitivity comparable to conventional PCR. Specifically, the implementation of the pincer heating scheme homogenises the temperature within a drop. The utilization of a µm-scale porous hydrophobic membrane suppresses the formation of bubbles under high temperatures. The design of a groove around the high-temperature zone effectively mitigates the temperature interference. The integration of a soluble sensor into the droplets provides an accurate and instant in-drop temperature sensing. We envision that the fast, robust, sensitive, and automatic DMF system will empower the POC testing for infectious diseases.

A Fully-Integrated Ambient RF Energy Harvesting System with 423-µW Output Power.

This paper proposes a 2.4-GHz fully-integrated single-frequency multi-channel RF energy harvesting (RFEH) system with increased harvested power density. The RFEH can produce an output power of ~423-µW in harvesting ambient RF energy. The front-end consists of an on-chip impedance matching network with a stacked rectifier concurrently matched to a 50 Ω input source. The circuit mitigates the "dead-zone" by enhancing the pumping efficiency, achieved through the increase of Vgs drivability of the proposed internal gate boosting 6-stage low-input voltage charge pump and the 5-stage shared-auxiliary-biasing ring-voltage-controlled-oscillator (VCO) integrated to improve the start-up. The RFEH system, simulated in 180-nm complementary metal-oxide-semiconductor (CMOS), occupies an active area of 1.02 mm2. Post-layout simulations show a peak power conversion efficiency(PCE) of 21.15%, driving a 3.3-kΩ load at an input power of 0 dBm and sensitivity of -14.1 dBm corresponding to an output voltage, Vout,RFEH of 1.25 V.

One-shot high-resolution melting curve analysis for KRAS point-mutation discrimination on a digital microfluidics platform.

Single-nucleotide polymorphism (SNP) plays a critical role in personalized medicine, forensics, pharmacogenetics, and disease diagnostics. Among different existing SNP genotyping techniques, melting curve analysis (MCA) becomes increasingly popular due to its high accuracy and straightforward procedures in extracting the melting temperature (Tm). Yet, its study on existing digital microfluidic (DMF) platforms has intrinsic limitations due to the temperature inhomogeneity within a thickened droplet during the on-chip rapid heating process. Although the utilization of an on-chip thermostat can regulate and monitor the dynamic melting process in real time, the limited Tm accuracy resulting from the insufficient system response time to accommodate the fast-melting evolution still poses a great challenge for precise MCA with high throughput. This work proposes a one-shot MCA on a DMF platform. The tailoring of a functional substrate with hierarchical micro/nano structure enables high-resolution patterning of pL-scale droplets. Specifically, the hydrothermal and photocatalysis treatment allows the functional substrate to exhibit a superwettability contrast of >170°, facilitating passive isolation of the pL-scale DNA sample into highly-resolved pL droplets above the 200 µm superhydrophilic patterns. This high-resolution MCA technique can successfully discriminate KRAS gene targets with single-nucleotide mutations in 3 seconds. The high accuracy and consistency in the acquired Tm when compared with off-chip results demonstrate its opportunities for near-patient diagnostics, precision medicines, genetic counseling, and prevention strategies on DMF platforms.

Cancer drug screening with an on-chip multi-drug dispenser in digital microfluidics.

Microfluidics has been the most promising platform for drug screening with a limited number of cells. However, convenient on-chip preparation of a wide range of drug concentrations remains a large challenge and has restricted wide acceptance of microfluidics in precision medicine. In this paper, we report a digital microfluidic system with an innovative control structure and chip design for on-chip drug dispensing to generate concentrations that span three to four orders of magnitude, enabling single drug or combinatorial multi-drug screening with simple electronic control. Specifically, we utilize droplet ejection from a drug drop sitting on a special electrode, named a drug dispenser, under high-voltage pulse actuation to deliver the desired amount of drugs to be picked up by a cell suspension drop driven by low-voltage sine wave actuation. Our proof-of-principle validation for this technique as a convenient single and multi-drug screening involved testing of the drug toxicity of two chemotherapeutics, cisplatin (Cis) and epirubicin (EP), towards MDA-MB-231 breast cancer cells and MCF-10A normal breast cells. The results are consistent with those screened based on traditional 96-well plates. These findings demonstrate the reliability of the drug screening system with an on-chip drug dispenser. This system with fewer cancer cells, less drug consumption, a small footprint, and high scalability with regard to concentration could pave the way for drug screening on biopsied primary tumor cells for precision medicine or any concentration-related research.

Temperature Tolerance Electric Cell-Substrate Impedance Sensing for Joint Assessment of Cell Viability and Vitality.

Evaluation of the cell health status is critical for drug screening and cell physiological activity investigations. The existing cell health assessment methods are solely devoted to the study of cell vitality or viability, leading to an incomplete evaluation. Herein, we report a convenient and robust method for the joint assessment of cell viability and vitality based on electric cell-substrate impedance sensing (ECIS) supplied with an environmental temperature control. The static value of electric cell-substrate impedance reflects the survival rate of cells, while the temperature tolerance of cells demonstrates the cell vitality. It was found that the cell vitality evaluated by the temperature tolerance of cells was independent of the initial cell numbers, rendering the proposed method easy to utilize in various applications. We compared the temperature tolerance ECIS method with the traditional trypan blue staining method, the methyl thiazolyl tetrazolium assay, and the direct impedance sensing method for joint evaluation of cell viability and vitality in drug screening. The temperature tolerance ECIS method showed comparable results but with a simpler protocol, faster results, and less dependence on the sample conditions. By providing both information on cell viability and cell vitality, the proposed temperature tolerance ECIS method would pave the way in building a simple and robust sensing system for cell health evaluation.

SARS-CoV-2 RNA Detection with Duplex-Specific Nuclease Signal Amplification.

The emergence of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a zoonotic pathogen, has led to the outbreak of coronavirus disease 2019 (COVID-19) pandemic and brought serious threats to public health worldwide. The gold standard method for SARS-CoV-2 detection requires both reverse transcription (RT) of the virus RNA to cDNA and then polymerase chain reaction (PCR) for the cDNA amplification, which involves multiple enzymes, multiple reactions and a complicated assay optimization process. Here, we developed a duplex-specific nuclease (DSN)-based signal amplification method for SARS-CoV-2 detection directly from the virus RNA utilizing two specific DNA probes. These specific DNA probes can hybridize to the target RNA at different locations in the nucleocapsid protein gene (N gene) of SARS-CoV-2 to form a DNA/RNA heteroduplex. DSN cleaves the DNA probe to release fluorescence, while leaving the RNA strand intact to be bound to another available probe molecule for further cleavage and fluorescent signal amplification. The optimized DSN amount, incubation temperature and incubation time were investigated in this work. Proof-of-principle SARS-CoV-2 detection was demonstrated with a detection sensitivity of 500 pM virus RNA. This simple, rapid, and direct RNA detection method is expected to provide a complementary method for the detection of viruses mutated at the PCR primer-binding regions for a more precise detection.

Turning on/off satellite droplet ejection for flexible sample delivery on digital microfluidics.

Digital microfluidics has the potential to minimize and automate reactions in biochemical labs. However, the complexity of drop manipulation and sample preparation on-chip has limited its incorporation into daily workflow. In this paper, we report a novel method for flexible sample delivery on digital microfluidics in a wide volume range spanning four orders of magnitude from picoliters to nanoliters. The method is based on the phenomenon of satellite droplet ejection, triggered by a sudden change in the strength of the electric field across a drop on a hydrophobic dielectric surface. By precisely modulating the actuation signal with convenient external electric controls, satellite droplet ejection can be turned on to dispense samples or turned off to transport picking-up drops. A pico-dosing design is presented and validated in this work to demonstrate the direct and flexible on-chip sample delivery. This approach could pave the way for the acceptance of microfluidics as a common platform for daily reactions to realize lab-on-a-chip.

Clip-to-release on amplification (CRoA): a novel DNA amplification enhancer on and off microfluidics.

Despite its high sensitivity, low cost, and high efficiency as a DNA amplification indicator with a yes/no answer, dsDNA-binding dye encounters incompatibility when used in microfluidic systems, resulting in problems such as false negative amplification results. Besides, its inhibition of amplification at high concentrations hinders its application both on-chip and off-chip. In this study, we propose a novel DNA amplification enhancer to counteract the drawbacks of dsDNA-binding dyes. It acts as a temporary reservoir for the free-floating dyes in solution and releases them on demand during the amplification process. Through this clip-to-release on amplification mechanism, the enhancer lowered the background fluorescence of sample droplets before amplification, enhanced the signal-to-background ratio of positive samples, and eliminated the false negative signal of on-chip PCR. Moreover, the enhancer increased the off-chip polymerase chain reaction (PCR) efficiency, boosted the fluorescence signal up to 10-fold, and made less nonspecific amplification product. All the factors affecting the enhancer's performance are investigated in detail, including its structure and concentration, and the types of dsDNA-binding dye used in the reaction. Finally, we demonstrated the broad application of the proposed amplification enhancer in various DNA amplification systems, for various genes, and on various amplification platforms. It would reignite the utilization of dsDNA dyes for wider applications in DNA analysis both on-chip and off-chip.

Portable NMR with Parallelism.

Portable NMR combining a permanent magnet and a complementary metal-oxide-semiconductor (CMOS) integrated circuit has recently emerged to offer the long desired online, on-demand, or in situ NMR analysis of small molecules for chemistry and biology. Here we take this cutting-edge technology to the next level by introducing parallelism to a state-of-the-art portable NMR platform to accelerate its experimental throughput, where NMR is notorious for inherently low throughput. With multiple (N) samples inside a single magnet, we perform simultaneous NMR analyses using a single silicon electronic chip, going beyond the traditional single-sample-per-magnet paradigm. We execute the parallel analyses via either time-interleaving or magnetic resonance imaging (MRI). In the time-interleaving method, the N samples occupy N separate NMR coils: we connect these N NMR coils to the single silicon chip one after another and repeat these sequential NMR scans. This time-interleaving is an effective parallelization, given a long recovery time of a single NMR scan. To demonstrate this time-interleaved parallelism, we use N = 2 for high-resolution multidimensional spectroscopy such as J-coupling resolved free induction decay spectroscopy and correlation spectroscopy (COSY) with the field homogeneity carefully optimized (<0.16 ppm) and N = 4 for multidimensional relaxometry such as diffusion-edited T2 mapping and T1-T2 correlation mapping, expediting the throughput by 2-4 times. In the MRI technique, the N samples (N = 18 in our demonstration) share 1 NMR coil connected to the single silicon chip and are imaged all at once multiple times, which reveals the relaxation time of all N samples simultaneously. This imaging-based approach accelerates the relaxation time measurement by 4.5 times, and it could be by 18 times if the signal-to-noise were not limited. Overall, this work demonstrates the first portable high-resolution multidimensional NMR with throughput-accelerating parallelism.

A digital microfluidic system with 3D microstructures for single-cell culture.

Despite the precise controllability of droplet samples in digital microfluidic (DMF) systems, their capability in isolating single cells for long-time culture is still limited: typically, only a few cells can be captured on an electrode. Although fabricating small-sized hydrophilic micropatches on an electrode aids single-cell capture, the actuation voltage for droplet transportation has to be significantly raised, resulting in a shorter lifetime for the DMF chip and a larger risk of damaging the cells. In this work, a DMF system with 3D microstructures engineered on-chip is proposed to form semi-closed micro-wells for efficient single-cell isolation and long-time culture. Our optimum results showed that approximately 20% of the micro-wells over a 30 × 30 array were occupied by isolated single cells. In addition, low-evaporation-temperature oil and surfactant aided the system in achieving a low droplet actuation voltage of 36V, which was 4 times lower than the typical 150 V, minimizing the potential damage to the cells in the droplets and to the DMF chip. To exemplify the technological advances, drug sensitivity tests were run in our DMF system to investigate the cell response of breast cancer cells (MDA-MB-231) and breast normal cells (MCF-10A) to a widely used chemotherapeutic drug, Cisplatin (Cis). The results on-chip were consistent with those screened in conventional 96-well plates. This novel, simple and robust single-cell trapping method has great potential in biological research at the single cell level.

A Novel and Robust Single-cell Trapping Method on Digital Microfluidics.

Due to cell heterogeneity, the differences among individual cells are averaged out in bulk analysis methods, especially in the analysis of primary tumor biopsy samples from patients. To deeply understand the cell-to-cell variation in a primary tumor, single-cell culture and analysis with limited amount of cells are in high demand. Microfluidics has been an optimum platform to address the issue given its small reaction volume requirements. Digital microfluidics, which utilizes an electric signal to manipulate individual droplets has shown promise in cell-culture with easy controls. In this work, we realize single cell trapping on digital microfluidic platform by fabricating 3D microstructures on-chip to form semi-closed micro-wells. With this design, 20% of 30 x 30 array can be occupied by isolated single cells. We also use a low evaporation silicon oil and a fluorinated surfactant to lower the droplet actuation voltage and prevent the drop from evaporation, while allowing cell respiration during the long term of culture (24 h). The main steps for single cell trapping on digital microfluidics, as illustrated in this protocol, include 3D microstructures design, 3D microstructures construction on chip and oil film with surfactant for single cell trapping on chip.

Author Correction: Drug screening of cancer cell lines and human primary tumors using droplet microfluidics.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

LampPort: a handheld digital microfluidic device for loop-mediated isothermal amplification (LAMP).

A major goal in the development of point-of-care (POC) devices is to build them as portable to provide a rapid and effective determination for disease pathogens. In nucleic acid testing, an optical detection system used to monitor the product of nucleic acid amplification has always been a bulky accessory. In this work, we developed a handheld, automatic and detection system-free thermal digital microfluidic (DMF) device for DNA detection by loop-mediated isothermal amplification (LAMP). Droplet manipulation and real-time temperature control systems were integrated into a handheld device. The control software could be installed into any tablet and communicate with the device via Bluetooth. In the experimentation, we loaded 2-µl samples with an electrowetting force into sandwich-structured DMF chips, thereby considerably reducing reagent consumptions. After an on-chip LAMP reaction, we added a highly concentrated SYBR Green I droplet and mixed it with a reaction droplet to enable product detection with the naked eye. This step prevented aerosol contamination by avoiding the exposure of the reaction droplet to the air. Using a blood parasite Trypanosoma brucei as a model system, this system showed similar results as a commercial thermal cycler and could detect 40 copies per reaction of the DNA target. This low-cost, compact device removed the bulky optical system for DNA detection, thus enabling it to be well suited for POC testing.

A digital microfluidic system for loop-mediated isothermal amplification and sequence specific pathogen detection.

A digital microfluidic (DMF) system has been developed for loop-mediated isothermal amplification (LAMP)-based pathogen nucleic acid detection using specific low melting temperature (Tm) Molecular Beacon DNA probes. A positive-temperature-coefficient heater with a temperature sensor for real-time thermal regulation was integrated into the control unit, which generated actuation signals for droplet manipulation. To enhance the specificity of the LAMP reaction, low-Tm Molecular Beacon probes were designed within the single-stranded loop structures on the LAMP reaction products. In the experiments, only 1 µL of LAMP reaction samples containing purified Trypanosoma brucei DNA were required, which represented over a 10x reduction of reagent consumption when comparing with the conventional off-chip LAMP. On-chip LAMP for unknown sample detection could be accomplished in 40 min with a detection limit of 10 copies/reaction. Also, we accomplished an on-chip melting curve analysis of the Molecular Beacon probe from 30 to 75 °C within 5 min, which was 3x faster than using a commercial qPCR machine. Discrimination of non-specific amplification and lower risk of aerosol contamination for on-chip LAMP also highlight the potential utilization of this system in clinical applications. The entire platform is open for further integration with sample preparation and fluorescence detection towards a total-micro-analysis system.

Drug screening of cancer cell lines and human primary tumors using droplet microfluidics.

Precision Medicine in Oncology requires tailoring of therapeutic strategies to individual cancer patients. Due to the limited quantity of tumor samples, this proves to be difficult, especially for early stage cancer patients whose tumors are small. In this study, we exploited a 2.4 × 2.4 centimeters polydimethylsiloxane (PDMS) based microfluidic chip which employed droplet microfluidics to conduct drug screens against suspended and adherent cancer cell lines, as well as cells dissociated from primary tumor of human patients. Single cells were dispersed in aqueous droplets and imaged within 24 hours of drug treatment to assess cell viability by ethidium homodimer 1 staining. Our results showed that 5 conditions could be screened for every 80,000 cells in one channel on our chip under current circumstances. Additionally, screening conditions have been adapted to both suspended and adherent cancer cells, giving versatility to potentially all types of cancers. Hence, this study provides a powerful tool for rapid, low-input drug screening of primary cancers within 24 hours after tumor resection from cancer patients. This paves the way for further technological advancement to cutting down sample size and increasing drug screening throughput in advent to personalized cancer therapy.