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.

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.

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.

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.

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.

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.

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.

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.

A 3D microblade structure for precise and parallel droplet splitting on digital microfluidic chips.

Existing digital microfluidic (DMF) chips exploit the electrowetting on dielectric (EWOD) force to perform droplet splitting. However, the current splitting methods are not flexible and the volume of the droplets suffers from a large variation. Herein, we propose a DMF chip featuring a 3D microblade structure to enhance the droplet-splitting performance. By exploiting the EWOD force for shaping and manipulating the mother droplet, we obtain an average dividing error of <2% in the volume of the daughter droplets for a number of fluids such as deionized water, DNA solutions and DNA-protein mixtures. Customized droplet splitting ratios of up to 20 : 80 are achieved by positioning the blade at the appropriate position. Additionally, by fabricating multiple 3D microblades on one electrode, two to five uniform daughter droplets can be generated simultaneously. Finally, by taking synthetic DNA targets and their corresponding molecular beacon probes as a model system, multiple potential pathogens that cause sepsis are detected rapidly on the 3D-blade-equipped DMF chip, rendering it as a promising tool for parallel diagnosis of diseases.

A Single-Chip Solar Energy Harvesting IC Using Integrated Photodiodes for Biomedical Implant Applications.

In this paper, an ultra-compact single-chip solar energy harvesting IC using on-chip solar cell for biomedical implant applications is presented. By employing an on-chip charge pump with parallel connected photodiodes, a 3.5 × efficiency improvement can be achieved when compared with the conventional stacked photodiode approach to boost the harvested voltage while preserving a single-chip solution. A photodiode-assisted dual startup circuit (PDSC) is also proposed to improve the area efficiency and increase the startup speed by 77%. By employing an auxiliary charge pump (AQP) using zero threshold voltage (ZVT) devices in parallel with the main charge pump, a low startup voltage of 0.25 V is obtained while minimizing the reversion loss. A 4 Vin gate drive voltage is utilized to reduce the conduction loss. Systematic charge pump and solar cell area optimization is also introduced to improve the energy harvesting efficiency. The proposed system is implemented in a standard 0.18- [Formula: see text] CMOS technology and occupies an active area of 1.54 [Formula: see text]. Measurement results show that the on-chip charge pump can achieve a maximum efficiency of 67%. With an incident power of 1.22 [Formula: see text] from a halogen light source, the proposed energy harvesting IC can deliver an output power of 1.65 [Formula: see text] at 64% charge pump efficiency. The chip prototype is also verified using in-vitro experiment.

CMOS biosensors for in vitro diagnosis - transducing mechanisms and applications.

Complementary metal oxide semiconductor (CMOS) technology enables low-cost and large-scale integration of transistors and physical sensing materials on tiny chips (e.g., <1 cm2), seamlessly combining the two key functions of biosensors: transducing and signal processing. Recent CMOS biosensors unified different transducing mechanisms (impedance, fluorescence, and nuclear spin) and readout electronics have demonstrated competitive sensitivity for in vitro diagnosis, such as detection of DNA (down to 10 aM), protein (down to 10 fM), or bacteria/cells (single cell). Herein, we detail the recent advances in CMOS biosensors, centering on their key principles, requisites, and applications. Together, these may contribute to the advancement of our healthcare system, which should be decentralized by broadly utilizing point-of-care diagnostic tools.

Sub-7-second genotyping of single-nucleotide polymorphism by high-resolution melting curve analysis on a thermal digital microfluidic device.

We developed a thermal digital microfluidic (T-DMF) device enabling ultrafast DNA melting curve analysis (MCA). Within 7 seconds, the T-DMF device succeeded in differentiating a melting point difference down to 1.6 °C with a variation of 0.3 °C in a tiny droplet sample (1.2 µL), which was 300 times faster and with 20 times less sample spending than the standard MCA (35 minutes, 25 µL) run in a commercial qPCR machine. Such a performance makes it possible for a rapid discrimination of single-nucleotide mutation relevant to prompt clinical decision-making. Also, aided by electronic intelligent control, the T-DMF device facilitates sample handling and pipelining in an automatic serial manner. An optimized oval-shaped thermal electrode is introduced to achieve high thermal uniformity. A device-sealing technique averts sample contamination and permits uninterrupted chemical/biological reactions. Simple fabrication using a single chromium layer fulfills both the thermal and typical transport electrode requirements. Capable of thermally modulating DNA samples with ultrafast MCA, this T-DMF device has the potential for a wide variety of life science analyses, especially for disease diagnosis and prognosis.

A palm-size µNMR relaxometer using a digital microfluidic device and a semiconductor transceiver for chemical/biological diagnosis.

Herein, we describe a micro-nuclear magnetic resonance (µNMR) relaxometer miniaturized to palm-size and electronically automated for multi-step and multi-sample chemical/biological diagnosis. The co-integration of microfluidic and microelectronic technologies enables an association between the droplet managements and µNMR assays inside a portable sub-Tesla magnet (1.2 kg, 0.46 Tesla). Targets in unprocessed biological samples, captured by specific probe-decorated magnetic nanoparticles (NPs), can be sequentially quantified by their spin-spin relaxation time (T2) via multiplexed µNMR screening. Distinct droplet samples are operated by a digital microfluidic device that electronically manages the electrowetting-on-dielectric effects over an electrode array. Each electrode (3.5 × 3.5 mm(2)) is scanned with capacitive sensing to locate the distinct droplet samples in real time. A cross-domain-optimized butterfly-coil-input semiconductor transceiver transduces between magnetic and electrical signals to/from a sub-10 µL droplet sample for high-sensitivity µNMR screening. A temperature logger senses the ambient temperature (0 to 40 °C) and a backend processor calibrates the working frequency for the transmitter to precisely excite the protons. In our experiments, the µNMR relaxometer quantifies avidin using biotinylated Iron NPs (Φ: 30 nm, [Fe]: 0.5 mM) with a sensitivity of 0.2 µM. Auto-handling and identification of two targets (avidin and water) are demonstrated and completed within 2.2 min. This µNMR relaxometer holds promise for combinatorial chemical/biological diagnostic protocols using closed-loop electronic automation.

NMR-DMF: a modular nuclear magnetic resonance-digital microfluidics system for biological assays.

We present a modular nuclear magnetic resonance-digital microfluidics (NMR-DMF) system as a portable diagnostic platform for miniaturized biological assays. With increasing number of combinations between designed probes and a specific target, NMR has become an accurate and rapid assay tool, which is capable of detecting particular kinds of proteins, DNAs, bacteria and cells with a customized probe quantitatively. Traditional sample operation (e.g., manipulation and mixing) relied heavily on human efforts. We herein propose a modular NMR-DMF system to allow the electronic automation of multi-step reaction-screening protocols. A figure-8 shaped coil is proposed to enlarge the usable inner space of a portable magnet by 4.16 times, generating a radio frequency (RF) excitation field in the planar direction. By electronically managing the electro-wetting-on-dielectric (EWOD) effects over an electrode array, preloaded droplets with the inclusion of biological constituents and targets can be programmed to mix and be guided to the detection site (3.5 × 3.5 mm(2)) for high-sensitivity NMR screening (static B field: 0.46 T, RF field: 1.43 mT per ampere), with the result (voltage signal) displayed in real-time. To show the system's utility, automated real-time identification of 100 pM of avidin in a 14 µL droplet was achieved. The system shows promise as a robust and portable diagnostic device for a wide variety of biological analyses and screening applications.