Lab on a Chip最新文献

Phenylketonuria (PKU) is characterized by an autosomal recessive mutation in the phenylalanine hydroxylase (PAH) gene. Impaired PAH enzyme activity leads to the accumulation of phenylalanine (Phe) and its metabolites in the bloodstream, which disrupts the central nervous system and causes psychomotor retardation. Early diagnosis of PKU is essential for timely intervention. Moreover, continuous monitoring of blood Phe levels is indispensable for prognosis, requiring a robust and reliable monitoring system. This study presents an automated lab-on-a-CD-based system for early diagnosis and monitoring of PKU treatment. This miniaturised system contains CD-shaped disposable cartridges, a mini centrifuge, and an electrochemical sensing unit. Modified screen-printed gold electrodes were used for the electrochemical measurements in cartridges. Electrode modification was conducted by electrochemical graphene oxide reduction and deposition on the electrode surface, which increased the sensitivity of the measurement 1.5 fold. The system used amperometric detection to measure Phe in the blood through oxidation of NAD⁺ to NADH by the enzyme phenylalanine dehydrogenase. The limit of detection (LOD), limit of quantification (LOQ), and sensitivity of the system were 0.0524, 0.1587 mg dL^-1 and 0.3338 μA mg^-1 dL, respectively, within the 0-20 mg dL^-1 measurement range (R² = 0.9955). The performance of the lab-on-a-CD system was compared to the gold standard HPLC method. The accuracy was 83.1% for HPLC and 84.1% for the lab-on-a-CD system. In conclusion, this study successfully developed a portable diagnostic device for rapid (under 20 min), accurate and highly sensitive detection of Phe in whole blood.

pH regulation of eukaryotic cells is of crucial importance and influences different mechanisms including chemical kinetics, buffer effects, metabolic activity, membrane transport and cell shape parameters. In this study, we develop a microfluidic system to rapidly and precisely control a continuous flow of ionic chemical species to acutely challenge the intracellular pH regulation mechanisms and confront predictive models. We monitor the intracellular pH dynamics in real-time using pH-sensitive fluorescence imaging and establish a robust mathematical tool to translate the fluorescence signals to pH values. By varying flow rate across the cells and duration for the rinsing process, we manage to tweak the dynamics of intracellular pH from a smooth recovery to either an overshooting state, where the pH goes excitedly to a maximum value before decreasing to a plateau, or an undershooting state, where the pH is unable to recover to ∼7. We believe our findings will provide more insight into intracellular regulatory mechanisms and promote the possibility of exploring cellular behavior in the presence of strong gradients or fast changes in homogeneous conditions.

Rapid and accurate molecular diagnostics are crucial for preventing the global spread of emerging infectious diseases. However, the current gold standard for nucleic acid detection, reverse transcription polymerase chain reaction (RT-PCR), relies heavily on traditional magnetic beads or silica membranes for nucleic acid extraction, resulting in several limitations, including time-consuming processes, the need for trained personnel, and complex equipment. Therefore, there is an urgent need for fully integrated nucleic acid detection technologies that are simple to operate, rapid, and highly sensitive to meet unmet clinical needs. In this study, we developed a novel, integrated microfluidic cartridge featuring a unique needle-plug/piston microvalve, which enables stable long-term reagent storage and flexible liquid handling for on-site nucleic acid analysis. Coupled with in situ tetra-primer recombinase polymerase amplification (tp-RPA), we achieved highly sensitive nucleic acid detection with a remarkable limit of detection of 20 copies per mL (0.02 copies per μL) and a short turnaround time of less than 30 minutes. To validate this assay, we tested 48 oropharyngeal swab samples. The positive detection rate reached 64.58% (31/48), significantly exceeding the approximately 50% positive detection rate of the traditional RT-PCR method. Furthermore, our assay demonstrated a 100% concordance rate with RT-PCR in detecting positive samples. Thus, we believe our microfluidic nucleic acid analysis system represents a promising approach for enabling rapid and ultrasensitive nucleic acid detection of pathogenic microorganisms in resource-limited settings and low-income areas.

Nucleic acid testing (NAT) is widely considered the gold standard in analytical fields, with applications spanning environmental monitoring, forensic science and clinical diagnostics, among others. However, its widespread use is often constrained by complicated assay procedures, the need for specialized equipment, and the complexity of reagent handling. In this study, we demonstrate a fully integrated 3D-printed biosensensing device employing a CRISPR/Cas12a-based dual-enzymatic mechanism for highly sensitive and user-friendly nucleic acid detection. A plastic probe stick was designed to host small-sized gold nanoparticles, enhancing enzyme labeling density. Alkaline phosphatase (ALP) was then conjugated via single-stranded DNA, requiring only a single enzyme substrate addition to generate a simple visual signal change. This approach eliminates the need for amplification or centrifugation steps, achieving a limit of detection (LOD) as low as 10 pM – among the highest sensitivities reported for amplification-free colorimetric nucleic acid detection. Furthermore, we developed a device that incorporates this probe stick, integrates all necessary reagents, and features a smartphone-compatible accessory for quantitative analysis. This allows end-users to perform visual or quantitative DNA analysis with simple operations, achieving a visual detection limit of approximately 100 pM, comparable to other CRISPR-based non-amplified nucleic acid detection methods. Additionally, the system successfully distinguished perfectly matched from mismatched nucleic acid sequences, demonstrating its specificity and versatility. Although certain design limitations affected the sensitivity of the integrated device compared to the probe stick alone, the simplicity and portability of this device make it a promising tool for rapid nucleic acid screening in clinical diagnostics, environmental monitoring, and food safety control. This study paves the way for the development of practical biosensors for point-of-care testing (POCT) applications.

In regular biosample cryopreservation operations, dropwise pipetting and continuous swirling are ordinarily needed to prevent cell damage (e.g. sudden osmotic change, toxicity and dissolution heat) caused by the high-concentration cryoprotectant (CPA) addition process. The following CPA removal process after freezing and rewarming also requires multiple sample transfer processes and manual work. In order to optimize the cryopreservation process, especially for trace sample preservation, here we present a microfluidic approach integrating CPA addition, sample storage, CPA removal and sample resuspension processes on a 30 × 30 × 4 mm³ three-layer chip. The sample solution could be added into CPA solution with pre-generated increasing concentration to decrease possible osmotic damage. Utilizing specially designed microfluidic structure and fluid field analysis, on-chip sample enrichment and CPA removal were achieved. A novel dead-end micro valve strategy with a simplified control module was applied and evaluated to assist on-chip mixing and sample pellet resuspension. The entire biosample cryopreservation process was also performed that verified the functions of the integrated microfluidic platform. Altogether, this developed platform could be an effective approach to realize automatic, all-in-one, low-damage cryopreservation operation.

Advancements in bulk and microfluidic emulsion methodologies have enabled highly efficient, high-throughput implementations of biochemical assays. Spray-based techniques offer rapid generation, droplet immobilization, and accessibility, but remain relatively underutilized, likely because they result in random and polydisperse droplets. However, the polydisperse characteristic can be leveraged; at sufficiently high droplet numbers, sequential sprays will generate mixed droplets which effectively populate a combinatorial space. In this paper, we present a method involving the sequential spraying and mixing of solutions encoded with fluorophores. This generates combinatorial droplets with quantifiable concentrations that can be imaged over time. To demonstrate the method's performance and utility, we use it to investigate synergistic and antagonistic pairwise antibiotic interactions.

Proteases, an important class of enzymes that cleave proteins and peptides, carry a wealth of potentially useful information. Devices to enable routine and cost effective measurement of their activity could find frequent use in clinical settings for medical diagnostics, as well as some industrial contexts such as detecting on-line biological contamination. In particular, devices that make use of readouts involving magnetic particles may offer distinct advantages for continuous sensing because material they release can be magnetically captured downstream and their readout is insensitive to optical properties of the sample. Bioassays based on giant magnetoresistance sensors that detect the binding or release of magnetic materials have been widely explored for these reasons, but they typically require expensive consumables. Here, we develop a simpler protease sensor based on inductive detection of particle release with pulsed magnetic fields, leveraging a design that incorporates both the pulse coil and gradiometer coils into a printed circuit board. Our fluidic chips are formed from casts of 3D printed molds, such that both the sensor and the consumable components could be relatively easy to mass produce. Using pulses ranging up to 10 s of mT, we show that our device has a limit of detection below 1 μg of iron and that its duty cycle can be varied to control temperature through Joule heating. By chemically functionalizing the glass surface of our fluidic chips with zwitterionic polymer and incorporating a PEG block co-polymer into the PDMS component, we are able to suppress the nonspecific binding of albumin by 7.8 times inside the chips. We demonstrate a layer-by-layer approach for covalently linking magnetic nanoparticles to the chips via cleavable peptide substrates. Finally, we observe the release of the magnetic particles from the chips under conditions of proteolytic cleavage and measure resulting changes in inductive signals, demonstrating a detection sensitivity for chymotrypsin in the hundreds of nM. The methods we establish here have the potential to aid progress toward sensors comprised of disposable fluidic chips measured by inexpensive detection devices that may one day facilitate ubiquitous protease activity monitoring.

Electrochemical impedance spectroscopy (EIS) serves as a non-invasive technique for assessing cell status, while mechanical stretching plays a pivotal role in stimulating cells to emulate their natural environment. Integrating these two domains enables the concurrent application of mechanical stimulation and EIS in a stretchable cell culture system. However, challenges arise from the difficulty in creating a durable and stable stretchable impedance electrode array. To overcome this limitation, we have developed a cell culture system integrated with a stretchable impedance electrode array (SIEA). This design enabled impedance monitoring during cell proliferation under mechanical stimulation over a long period of time due to the high mechanical durability and electrochemical stability of the SIEA. Our evaluation also involved testing the cytotoxicity of doxorubicin on H9C2 cells directly on the SIEA. The results underscore the significant potential of our SIEA device as an effective tool for monitoring cells subjected to mechanical stimulation and as a platform for drug toxicity testing.

The utilization of acoustic fields offers a contactless approach for microparticle manipulation in a miniaturized system, and plays a significant role in medicine, biology, chemistry, and engineering. Due to the acoustic radiation force arising from the scattering of the acoustic waves, small particles in the Rayleigh scattering range can be trapped, whilst their impact on the acoustic field is negligible. Manipulating larger particles in the Mie scattering regime is challenging due to the diverse scattering modes, which impacts the local acoustic field. The rapid movement of free-moving Mie scatterers in an acoustic standing wave field makes it difficult to study the interaction between a sound field and a Mie scatterer in an engineering context. Here, a combined approach that integrates theoretical analysis and experimental investigation was developed to explore the influence of a Mie scatterer on the acoustic field by fabricating an acoustic trapping device featuring a fixed Mie scatterer at its center. We demonstrate that an insonified Mie scatterer can operate as an acoustic emitter in water, enabling dynamic and versatile modulation of the total acoustic field. Such a scatterer can interact with one or multiple incident propagating acoustic waves, leading to the generation of a localized standing wave field in the vicinity of the scatterer. This local field can be controlled by the relative location of the scatterer with respect to the incident field leading to control over the transformation from an incident 1D acoustic field into a 2D acoustic field. This control paves the way for localized and multi-scale micro-object manipulation.

Acoustic waves provide an effective method for object manipulation in microfluidics, often requiring high-frequency ultrasound in the megahertz range when directly handling microsized objects, which can be costly. Micro-air-bubbles in water offer a solution toward low-cost technologies using low-frequency acoustic waves. Owing to their high compressibility and low elastic modulus, these bubbles can exhibit significant expansion and contraction in response to even kilohertz acoustic waves, leading to resonances with frequencies determined and tuned by air-bubble size. The resonances amplify vibrational amplitude and generate localized turbulence, enabling selective, non-invasive, and high-precision manipulation of microsized objects. However, conventional bubble formation relies on the shear force of the liquid flow and bubble surface tension, facing challenges of instability and random vibration that can impair manipulation precision and performance. To address these issues, we propose a coupled vibration structure with 3D-printed circular microsized air holes encapsulated by a PDMS film. These airholes act as artificial micro-air-bubbles, with their expansion and contraction stabilized by acoustic hard boundaries. The PDMS film further regulates vibration modes through the interaction between air movement and the film's vibration, eliminating randomness. Compared to conventional air-bubbles held by surface tension, these artificial air-bubbles are mechanically stable, allowing for enhanced gas volume changes and stronger forces for object manipulation. We experimentally confirm the stable vibration modes and their frequency-dependent behavior using laser Doppler vibrometry. Precise aggregation, rotation, and separation of micro-objects are demonstrated by adjusting the film's vibration mode. Furthermore, we propose a metasurface design featuring a multi-size microbubble array for frequency-selective manipulation, enabling flexible control of sample trajectory by changing the exciting frequency of an embedded piezoelectric transducer. Our low-frequency acoustic metasurface device offers a versatile, cost-effective solution for drug screening and automated sample handling.