Volatile carbonyl metabolites analysis of nanoparticle exposed lung cells in an organ-on-a-chip system.

The evaluation of nanoparticles (NPs) cytotoxicity is crucial for advancing nanotechnology and assessing environmental pollution. However, existing methods for NPs cytotoxicity evaluation suffer from limited accuracy and inadequate information content. In the study, we developed a novel detection platform that enables the identification of cellular carbonyl metabolites at the organ level. The platform is integrated with a cell co-culture lung organ chip (LOC) and a micropillar concentrator. Notably, our work represents the successful measurement of the amounts of cellular metabolites on LOC system. The volatile carbonyl metabolites (VCMs) generated by cells exposure to various types of NPs with different concentrations were captured and detected by high-resolution mass spectrometry (MS). Compared with conventional cell viability and reactive oxygen species (ROS) analysis, our method discerns the toxicological impact of NPs at low concentrations by analyzed VCM at levels as low as ppb level. The LOC system based metabolic gas detection confirmed that low concentrations of NPs have a toxic effect on the cell model, which was not reflected in the fluorescence detection, and the effect of NP material is more significant than the size effect. Furthermore, this method can distinguish different NPs acting on cell models through cluster analysis of multiple VCMs.

Antibody responses to SARS-CoV-2 Omicron infection in patients with hematological malignancies: A multicenter, prospective cohort study.

Little is known about antibody responses to natural Omicron infection and the risk factors for poor responders in patients with hematological malignancies (HM). We conducted a multicenter, prospective cohort study during the latest Omicron wave in Chongqing, China, aiming to compare the antibody responses, as assessed by IgG levels of anti-receptor binding domain of spike protein (anti-S-RBD), to Omicron infection in the HM cohort (HMC) with healthy control cohort (HCC), and solid cancer cohort (SCC). In addition, we intend to explore the risk factors for poor responders in the HMC. Among the 466 HM patients in this cohort, the seroconversion rate was 92.7%, no statistically difference compared with HCC (98.2%, p = 0.0513) or SCC (100%, p = 0.1363). The median anti-S-RBD IgG titer was 29.9 ng/mL, significantly lower than that of HCC (46.9 ng/mL, p < 0.0001) or SCC (46.2 ng/mL, p < 0.0001). Risk factors associated with nonseroconversion included no COVID-19 vaccination history (odds ratio [OR] = 4.58, 95% confidence interval [CI]: 1.75-12.00, p = 0.002), clinical course of COVID-19 ≤ 7 days (OR = 2.86, 95% CI: 1.31-6.25, p = 0.008) and severe B-cell reduction (0-10/µL) (OR = 3.22, 95% CI: 1.32-7.88, p = 0.010). Risk factors associated with low anti-S-RBD IgG titer were clinical course of COVID-19 ≤ 7 days (OR = 2.58, 95% CI: 1.59-4.18, p < 0.001) and severe B-cell reduction (0-10/µL) (OR = 2.87, 95% CI: 1.57-5.24, p < 0.001). This study reveals a poor antibody responses to Omicron (BA.5.2.48) infection in HM patients and identified risk factors for poor responders. Highlights that HM patients, especially those with these risk factors, may be susceptible to SARS-CoV-2 reinfection, and the postinfection vaccination strategies for these patients should be tailored. Clinical trial: ChiCTR2300071830.

piRT-IFC: Physics-informed real-time impedance flow cytometry for the characterization of cellular intrinsic electrical properties.

Real-time transformation was important for the practical implementation of impedance flow cytometry. The major obstacle was the time-consuming step of translating raw data to cellular intrinsic electrical properties (e.g., specific membrane capacitance Csm and cytoplasm conductivity σcyto). Although optimization strategies such as neural network-aided strategies were recently reported to provide an impressive boost to the translation process, simultaneously achieving high speed, accuracy, and generalization capability is still challenging. To this end, we proposed a fast parallel physical fitting solver that could characterize single cells' Csm and σcyto within 0.62 ms/cell without any data preacquisition or pretraining requirements. We achieved the 27000-fold acceleration without loss of accuracy compared with the traditional solver. Based on the solver, we implemented physics-informed real-time impedance flow cytometry (piRT-IFC), which was able to characterize up to 100,902 cells' Csm and σcyto within 50 min in a real-time manner. Compared to the fully connected neural network (FCNN) predictor, the proposed real-time solver showed comparable processing speed but higher accuracy. Furthermore, we used a neutrophil degranulation cell model to represent tasks to test unfamiliar samples without data for pretraining. After being treated with cytochalasin B and N-Formyl-Met-Leu-Phe, HL-60 cells underwent dynamic degranulation processes, and we characterized cell's Csm and σcyto using piRT-IFC. Compared to the results from our solver, accuracy loss was observed in the results predicted by the FCNN, revealing the advantages of high speed, accuracy, and generalizability of the proposed piRT-IFC.

Dynamic Detection of Specific Membrane Capacitance and Cytoplasmic Resistance of Neutrophils After Ischemic Stroke.

Peripheral blood is the most readily available resource for stroke patient prognosis, but there is a lack of methods to detect dynamic changes of neutrophils in peripheral blood that can be used in the clinic. Herein, we developed a procedure to characterize dynamic changes of neutrophils based on their electrical properties in rats after transient middle cerebral artery occlusion (MCAO). We characterized the specific membrane capacitance (Csm) and cytoplasmic resistance (σcyto) of approximately 27,600 neutrophils from MCAO rats 24 h after ischemia/reperfusion. We found that the Csm and σcyto of neutrophils in the MCAO group were significantly higher compared to the sham group. Furthermore, we observed a monotonically upward shift in neutrophil Csm in the MCAO group during the four 5-minute test cycles. Our findings suggest that the dynamic changes of cellular electrical properties could reflect neutrophil activity and serve as a prognostic indicator for ischemic stroke in the clinical setting.

From the teapot effect to tap-triggered self-wetting: a 3D self-driving sieve for whole blood filtration.

Achieving passive microparticle filtration with micropore membranes is challenging due to the capillary pinning effect of the membranes. Inspired by the teapot effect that occurs when liquid (tea) is poured from a teapot spout, we proposed a tap-triggered self-wetting strategy and utilized the method with a 3D sieve to filter rare cells. First, a 3D-printed polymer tap-trigger microstructure was implemented. As a result, the 3 µm micropore membrane gating threshold (the pressure needed to open the micropores) was lowered from above 3000 to 80 Pa by the tap-trigger microstructure that facilated the liquid leakage and spreading to self-wet more membrane area in a positive feedback loop. Then, we implemented a 3D cone-shaped cell sieve with tap-trigger microstructures. Driven by gravity, the sieve performed at a high throughput above 20 mL/min (DPBS), while the micropore size and porosity were 3 µm and 14.1%, respectively. We further filtered leukocytes from whole blood samples with the proposed new 3D sieve, and the method was compared with the traditional method of leukocyte isolation by chemically removing red blood cells. The device exhibited comparable leukocyte purity but a higher platelet removal rate and lower leukocyte simulation level, facilitating downstream single-cell analysis. The key results indicated that the tap-triggered self-wetting strategy could significantly improve the performance of passive microparticle filtration.

A parylene-mediated plasmonic-photonic hybrid fiber-optic sensor and its instrumentation for miniaturized and self-referenced biosensing.

With the development of advanced nanofabrication techniques over the past decades, different nanostructure-based plasmonic fiber-optic sensors have been developed and have presented a low limit of detection for various biomolecules. However, owing to both the dependence on complex equipment and the trade-off between the fabrication cost and sensing performance, nanostructured plasmonic fiber-optic sensors are rarely used outside laboratories. To facilitate wider application of the plasmonic fiber-optic sensors, a parylene-mediated hybrid plasmonic-photonic cavity-based sensor was developed. Compared with a similar plasmonic sensor which only works in the plasmonic mode, the proposed hybrid sensor shows a higher reproducibility (CV < 2.5%) due to its resistance to fabrication variations. Meanwhile, a self-referenced detection mechanism and a novel miniaturized system were developed to adapt to the hybrid resonance sensor. The entire system only has a weight of 263 g, and a size of 12 cm × 10 cm × 8 cm, and is especially suitable for outdoor applications in a handheld manner. In experiments, a high refractive index sensitivity of 3.148 RIU-1 and real-time biomolecule monitoring at nanomolar concentrations were achieved by the proposed system, further confirming the potential of the miniaturized system as a candidate for point-of-care health diagnostics outside laboratories.

Real-time measurement of the trans-epithelial electrical resistance in an organ-on-a-chip during cell proliferation.

The trans-epithelial electrical resistance (TEER) is widely used to quantitatively evaluate cellular barrier function at the organ level in vitro. The measurement of the TEER in organ-on-chips (organ chips) plays a significant role in medical and pharmacological research. However, due to the limitation of the electrical equivalent model for organ chips, the existing TEER measurements usually neglect the changes of the TEER during cell proliferation, resulting in the low accuracy of the measurements. Here, we proposed a new whole-region model of the TEER and developed a real-time TEER measurement system that contains an organ chip with a plate electrode. A whole region circuit model considering the impedance of the non-cell covered region was also established, which enables TEER measurements to be independent of the changes in the cell covered region. The impedance of the non-cell covered region is here attributed to the resistance of the porous membrane. By combining the real-time measurement system and the whole region model, subtle changes in cellular activity during the proliferation stage were measured continuously every 6 minutes and a more sensitive TEER response was obtained. Furthermore, the TEER measurement accuracy was also verified by the real-time measurement of the TEER with stimulation using the permeability enhancer ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA). The obtained results indicated that the new proposed whole region model and the real-time measurement system have higher accuracy and greater sensitivity than the traditional model.

Long-term efficacy and safety of programmed death-1 (PD-1) antibody alone in relapsed/refractory human immunodeficiency virus-associated Hodgkin lymphoma.

We report a young patient initially diagnosed with human immunodeficiency virus (HIV)-associated Hodgkin lymphoma (HL), and received six cycles of ABVD chemotherapy regimens and involvement field irradiation therapy. However, the disease progressed after two months later, and then received second line GDP regimen. Unfortunately, after five cycles of GDP, the patient progression disease (PD) again. The patient was then offered sintilimab alone. After 8 cycles, the patient received complete response (CR) and no 3/4 grade toxicity. Currently, at a follow-up period of four years, he is still alive with CR and no lymphoma-related symptoms. This case demonstrates the feasibility of sintilimab antibody in relapsed/refractory HIV-associated Hodgkin lymphoma.

A 3D Capillary-Driven Multi-Micropore Membrane-Based Trigger Valve for Multi-Step Biochemical Reaction.

Point-of-care testing (POCT) techniques based on microfluidic devices enabled rapid and accurate tests on-site, playing an increasingly important role in public health. As the critical component of capillary-driven microfluidic devices for POCT use, the capillary microfluidic valve could schedule multi-step biochemical operations, potentially being used for broader complex POCT tasks. However, owing to the reciprocal relationship between the capillary force and aperture in single-pore microchannels, it was challenging to achieve a high gating threshold and high operable liquid volume simultaneously with existing 2D capillary trigger valves. This paper proposed a 3D capillary-driven multi-microporous membrane-based trigger valve to address the issue. Taking advantage of the high gating threshold determined by micropores and the self-driven capillary channel, a 3D trigger valve composed of a microporous membrane for valving and a wedge-shaped capillary channel for flow pumping was implemented. Utilizing the capillary pinning effect of the multi-micropore membrane, the liquid above the membrane could be triggered by putting the drainage agent into the wedge-shaped capillary channel to wet the underside of the membrane, and it could also be cut off by taking away the agent. After theoretical analysis and performance characterizations, the 3D trigger valve performed a high gating threshold (above 1000 Pa) and high trigger efficiency with an operable liquid volume above 150 µL and a trigger-to-drain time below 6 s. Furthermore, the retention and trigger states of the valve could be switched for repeatable triggering for three cycles within 5 min. Finally, the microbead-based immunoreaction and live cell staining applications verified the valve's ability to perform multi-step operations. The above results showed that the proposed 3D trigger valve could be expected to play a part in wide-ranging POCT application scenarios.

Microsphere mediated exosome isolation and ultra-sensitive detection on a dielectrophoresis integrated microfluidic device.

Tumor-derived exosomes have been recognized as potential biomarkers for cancer diagnosis because they are actively involved in cancer progression and metastasis. However, progress in practical exosome analysis is still slow due to the limitation in exosome isolation and detection. The development of microfluidic devices has provided a promising analytical platform compared with traditional methods. In this study, we develop an exosome isolation and detection method based on a microfluidic device (ExoDEP-chip), which realized microsphere mediated dielectrophoretic isolation and immunoaffinity detection. Exosomes were firstly isolated by binding to antibodies pre-immobilized on the polystyrene (PS) microsphere surface and were further detected using fluorescently labeled antibodies by fluorescence microscopy. Single microspheres were then trapped into single microwells under the DEP force in the ExoDEP-chip. A wide range from 1.4 × 103 to 1.4 × 108 exosomes per mL with a detection limit of 193 exosomes per mL was obtained. Through monitoring five proteins (CD81, CEA, EpCAM, CD147, and AFP) of exosomes from three different cell lines (A549, HEK293, and HepG2), a significant difference in marker expression levels was observed in different cell lines. Therefore, this method has good prospects in exosome-based tumor marker detection and cancer diagnosis.

Characterization of porcine sapelovirus prevalent in western Jiangxi, China.

BACKGROUND: Porcine sapelovirus (PSV) infection can lead severe polioencephalomyelitis with high morbidity and mortality, which result in significant economic losses. Infection with the PSV is believed to be common yet limited information is available on the prevalence and molecular characterization of PSV in China. Therefore, the objective of this study was to characterize the prevalence and genome of PSV strains identified in the western Jiangxi province of China. RESULTS: A high specificity and sensitivity SYBR Green I-based RT-PCR method for PSV detection was developed. Two hundred and ninety four fecal samples were collected from December 2018 to March 2019 in 4 farms. An overall PSV-positivity rate of 11.22% (33/294) was detected with the real-time RT-PCR method, and a high infection rate and viral load of PSV were found in nursery pigs. In total, complete VP1 gene sequences of 11 PSV strains (PSV-YCs) were obtained. Homology comparisons of the VP1 gene of the 11 PSV-YCs with previously reported PSVs revealed nucleotide sequence identities ranging from 63% to 96.8%, and deduced amino acid sequence identities from 61.4% to 99.7%. Phylogenetic analyses based on the VP1 gene exhibited 2 main clades corresponding to PSV-1 and PSV-2, and all PSV-YCs prevalent in western Jiangxi belonged to the traditional genotype (PSV-1). In addition, the pairwise distances of VP1 gene sequences between PSV-YCs ranged from 0.009 to 0.198, which indicating that substantial genetic diversity among the PSVs in western Jiangxi. CONCLUSIONS: To the authors' knowledge, this is the first description of PSV in the Jiangxi province pig herds in China, and it is crucial to understand the epidemiology of the viruses in China. The results also provide an important theoretical foundation for diagnosis and early warning of epidemic diseases caused by PSVs prevailing in this region.

Convex-Meniscus-Assisted Self-Assembly at the Air/Water Interface to Prepare a Wafer-Scale Colloidal Monolayer Without Overlap.

Self-assembly at the air/water interface (AWI) has proven to be an efficient strategy for fabricating two-dimensional (2D) colloidal monolayers, which was widely used as the template for nanosphere lithography in nanophononics, optofluidics, and solar cell studies. However, the monolayers fabricated at the AWI usually suffer from a small domain area and quasi-double layer structure caused by submerged particles. To overcome this, we proposed an improved protocol to prepare 2D colloidal monolayers free of overlapping nanospheres at the AWI. Utilizing the stable suspension infusion to the water surface, a convex meniscus, whose height is related to viscous force, was formed adjoining the three-phase boundary. As a result of the resistance of the convex meniscus, the polystyrene nanospheres in the initial suspension directly self-assembled into a preliminary monolayer, which proved effective in preventing nanospheres' sinking and increasing the colloidal crystal domain size. An optimal parameter for transferring the monolayer was also developed based on the numerical simulation results. Finally, a wafer-scale monolayer, covered with less than one nanosphere per 100 µm × 100 µm area, was achieved on the desired substrate with an average domain size attaining centimeter scale. The high-quality 2D colloidal crystal may further promote the application of nanosphere lithography, especially in the fields that require a defect-free template.

Single-Cell Electroporation with Real-Time Impedance Assessment Using a Constriction Microchannel.

The electroporation system can serve as a tool for the intracellular delivery of foreign cargos. However, this technique is presently limited by the inaccurate electric field applied to the single cells and lack of a real-time electroporation metrics subsystem. Here, we reported a microfluidic system for precise and rapid single-cell electroporation and simultaneous impedance monitoring in a constriction microchannel. When single cells (A549) were continuously passing through the constriction microchannel, a localized high electric field was applied on the cell membrane, which resulted in highly efficient (up to 96.6%) electroporation. During a single cell entering the constriction channel, an abrupt impedance drop was noticed and demonstrated to be correlated with the occurrence of electroporation. Besides, while the cell was moving in the constriction channel, the stabilized impedance showed the capability to quantify the electroporation extent. The correspondence of the impedance variation and electroporation was validated by the intracellular delivery of the fluorescence indicator (propidium iodide). Based on the obtained results, this system is capable of precise control of electroporation and real-time, label-free impedance assessment, providing a potential tool for intracellular delivery and other biomedical applications.

Nanotechnology-Assisted Isolation and Analysis of Circulating Tumor Cells on Microfluidic Devices.

Circulating tumor cells (CTCs), a type of cancer cell that spreads from primary tumors into human peripheral blood and are considered as a new biomarker of cancer liquid biopsy. It provides the direction for understanding the biology of cancer metastasis and progression. Isolation and analysis of CTCs offer the possibility for early cancer detection and dynamic prognosis monitoring. The extremely low quantity and high heterogeneity of CTCs are the major challenges for the application of CTCs in liquid biopsy. There have been significant research endeavors to develop efficient and reliable approaches to CTC isolation and analysis in the past few decades. With the advancement of microfabrication and nanomaterials, a variety of approaches have now emerged for CTC isolation and analysis on microfluidic platforms combined with nanotechnology. These new approaches show advantages in terms of cell capture efficiency, purity, detection sensitivity and specificity. This review focuses on recent progress in the field of nanotechnology-assisted microfluidics for CTC isolation and detection. Firstly, CTC isolation approaches using nanomaterial-based microfluidic devices are summarized and discussed. The different strategies for CTC release from the devices are specifically outlined. In addition, existing nanotechnology-assisted methods for CTC downstream analysis are summarized. Some perspectives are discussed on the challenges of current methods for CTC studies and promising research directions.

Wafer-level fabrication of 3D nanoparticles assembled nanopillars and click chemistry modification for sensitive SERS detection of trace carbonyl compounds.

In this work, we develop a new method for fabricating wafer-level gold nanoparticles covered silicon nanopillars (SNPs) combined with surface chemical modification to detect trace level carbonyl compounds based on surface-enhanced Raman scattering (SERS) technique. The SNPs are fabricated with an etching process using nano masks synthesized in oxygen-plasma bombardment of photoresist, and further deposited with gold nanoparticles on the surface, thus forming a 3D 'particles on pillars' nanostructure for sensitive SERS detection. The enhancement factor (EF) of the devices for R6G detection can achieve 1.56 × 106 times compared with a flat Si substrate. We also developed an oximation click chemistry reaction procedure by chemically modifying the nanostructures with aminooxy dodecane thiol (ADT) self-assemble modification. The chip is further integrated with a polydimethylsiloxane (PDMS) microfluidic chamber, which allows fast and convenient detection of trace carbonyl compounds in liquid samples. The SERS detection capability was demonstrated by the dropwise addition of fluorescent carbonyl compounds before and after elution. Furthermore, the device was proved with high surface consistency(<70%) for repeated measurement, which has the potential for ppb(parts per billion) level concentration of carbonyl compounds detection.

Identification of novel genotypes belonging to the species Teschovirus A from indigenous pigs in western Jiangxi, China.

Teschovirus A is currently the sole species in the genus Teschovirus, whose members are divided into 13 subtypes: porcine teschovirus (PTV) 1-13. However, recent discoveries of novel PTV genotypes have suggested that a new species, "Teschovirus B", should be established. Here, we have identified six of the 19 known genotypes and two novel genotypes (PTV 17-18), revealing the high genetic diversity of the PTV subpopulation in indigenous pigs of western Jiangxi, China. Moreover, we determined the nearly complete genome sequences of PTV 17-SG9 and PTV 18-SG10. Together with PTV 1-13, these novel genotypes were confirmed to be members of the species Teschovirus A based on phylogenetic and genetic divergence analysis. Consequently, the species Teschovirus A now includes at least 15 PTV genotypes.

Novel species of Teschovirus B comprises at least three distinct evolutionary genotypes.

Conventionally, Porcine teschovirus (PTV) consists of 13 genotypes (PTV 1-13, which belong to Teschovirus A); however, several novel members including PTV 14-22 have been discovered recently. PTV 21 was identified as a novel Teschovirus species named Teschovirus B. In this study, almost all 22 reported PTV genotypes except PTV 6, 7, 12 and 20 were identified in the pig populations of western Jiangxi, China, which reflects the high genotype diversity. Moreover, to the best of our knowledge, the nearly complete genome of PTV 22-JiangX1 was first sequenced in the present study. The homology comparison of the polyprotein genes showed that PTV 22-JiangX1 shared a relatively high nucleotide and deduced amino acid sequence identities ranging from 78.3% to 82.0% and 84.6% to 89.3%, respectively, with PTV 19 and 21. Additionally, the PTV strain of JiangX1 represents genotype 22 with the PTV 19, and 21 strains proved to be members of Teschovirus B based on the phylogenetic and evolutionary divergence analyses. Finally, we determined that the novel Teschovirus B species comprises at least three PTV genotypes.

Microfabrication of Micropore Array for Cell Separation and Cell Assay.

Micropore arrays have attracted a substantial amount of attention due to their strong capability to separate specific cell types, such as rare tumor cells, from a heterogeneous sample and to perform cell assays on a single cell level. Micropore array filtration has been widely used in rare cell type separation because of its potential for a high sample throughput, which is a key parameter for practical clinical applications. However, most of the present micropore arrays suffer from a low throughput, resulting from a low porosity. Therefore, a robust microfabrication process for high-porosity micropore arrays is urgently demanded. This study investigated four microfabrication processes for micropore array preparation in parallel. The results revealed that the Parylene-C molding technique with a silicon micropillar array as the template is the optimized strategy for the robust preparation of a large-area and high-porosity micropore array, along with a high size controllability. The Parylene-C molding technique is compatible with the traditional micromechanical system (MEMS) process and ready for scale-up manufacture. The prepared Parylene-C micropore array is promising for various applications, such as rare tumor cell separation and cell assays in liquid biopsy for cancer precision medicine.

Enhanced parylene-C fluorescence as a visual marker for neuronal electrophysiology applications.

Parylene-C is a popular polymer material in biomedical applications, with excellent physicochemical properties and microfabrication capability. Like many aromatic polymers, parylene-C also has autofluorescence, which was usually taken as a negative background noise in biomedical detection studies. However, the fluorescence intensity of thin-film (<1 µm) parylene-C was relatively weak, which may be a big limitation in visualization. In this work, we reported a simple annealing method to significantly enhance the fluorescence and achieve sufficient intensity as a visual marker. We studied the behaviors and mechanisms of the enhanced parylene-C fluorescence, then verified the feasibility and reliability of parylene-C for preparing fluorescent pipettes in targeted neuronal electrophysiology, where fluorescent guidance was strongly needed. The powerful parylene-C fabrication technique enables a precisely-controlled conformal coating along with a mass production capability, which further resulted in high-quality electrophysiological recordings of both cultured hippocampal neurons and acute hippocampal brain slices. Moreover, the enhanced parylene-C fluorescence can also be applied in more general biological operations, such as designable fluorescent micro-patterns for visualization in broader biomedical fields.

SF6 Optimized O2 Plasma Etching of Parylene C.

Parylene C is a widely used polymer material in microfabrication because of its excellent properties such as chemical inertness, biocompatibility and flexibility. It has been commonly adopted as a structural material for a variety of microfluidics and bio-MEMS (micro-electro-mechanical system) applications. However, it is still difficult to achieve a controllable Parylene C pattern, especially on film thicker than a couple of micrometers. Here, we proposed an SF6 optimized O2 plasma etching (SOOE) of Parylene C, with titanium as the etching mask. Without the SF6, noticeable nanoforest residuals were found on the O2 plasma etched Parylene C film, which was supposed to arise from the micro-masking effect of the sputtered titanium metal mask. By introducing a 5-sccm SF6 flow, the residuals were effectively removed during the O2 plasma etching. This optimized etching strategy achieved a 10 µm-thick Parylene C etching with the feature size down to 2 µm. The advanced SOOE recipes will further facilitate the controllable fabrication of Parylene C microstructures for broader applications.