A Novel Rolling Circle Amplification-Based Detection of SARS-CoV-2 with Multi-Region Padlock Hybridization.

SARS-CoV-2 has remained a global health burden, primarily due to the continuous evolution of different mutant strains. These mutations present challenges to the detection of the virus, as the target genes of qPCR, the standard diagnostic method, may possess sequence alterations. In this study, we develop an isothermal one-step detection method using rolling circle amplification (RCA) for SARS-CoV-2. This novel strategy utilizes a multi-padlock (MP-RCA) approach to detect viral-RNA via a simplified procedure with the reliable detection of mutated strains over other procedures. We designed 40 padlock-based probes to target different sequences across the SARS-CoV-2 genome. We established an optimal one-step isothermal reaction protocol utilizing a fluorescent output detected via a plate reader to test a variety of padlock combinations. This method was tested on RNA samples collected from nasal swabs and validated via PCR. S-gene target failure (SGTF)-mutated strains of SARS-CoV-2 were included. We demonstrated that the sensitivity of our assay was linearly proportional to the number of padlock probes used. With the 40-padlock combination the MP-RCA assay was able to correctly detect 45 out 55 positive samples (81.8% efficiency). This included 10 samples with SGTF mutations which we were able to detect as positive with 100% efficiency. We found that the MP-RCA approach improves the sensitivity of the MP-RCA assay, and critically, allows for the detection of SARS-CoV-2 variants with SGTF. Our method offers the simplicity of the reaction and requires basic equipment compared to standard qPCR. This method provides an alternative approach to overcome the challenges of detecting SARS-CoV-2 and other rapidly mutating viruses.

Droplet microfluidics for functional temporal analysis and cell recovery on demand using microvalves: application in immunotherapies for cancer.

Most common methods of cellular analysis employ the top-down approach (investigating proteomics or genomics directly), thereby destroying the cell, which does not allow the possibility of using the same cell to correlate genomics with functional assays. Herein we describe an approach for single-cell tools that serve as a bottom-up approach. Our technology allows functional phenotyping to be conducted by observing the cytotoxicity of cells and then probe the underlying biology. We have developed a droplet microfluidic device capable of trapping droplets in the array and releasing the droplet of interest selectively using microvalves. Each droplet in the array encapsulates natural killer cells (NK cells) and tumour cells for real-time monitoring of burst kinetics and spatial coordination during killing by single NK cells. Finally, we use the microvalve actuation to selectively release droplets with the desired functional phenotype such as for fast and serial killing of target tumour cells by NK cells. From this perspective, our device allows for investigating first interactions and real-time monitoring of kinetics and later cell recovery on demand for single-cell omic analysis such as single-cell RNA sequencing (scRNA), which to date, is primarily based on in-depth analyses of the entire transcriptome of a relatively low number of cells.

Rolling Circle Amplification-based Copper Nanoparticle Synthesis on Cyclic Olefin Copolymer Substrate and Its Application in Aptasensor.

This study aimed to develop a label-free fluorescent aptasensor for the detection of diazinon (DZN) on a cyclic olefin copolymer (COC) substrate. The aptasensor design was based on rolling circle amplification (RCA) technology and the use of self-assembled copper nanoparticles (CuNPs). A dual-function (DF) probe, capable of binding to circular DNA and an aptamer, was designed and immobilized on a COC-bottom 96-well plate. An aptamer was used for selective recognition of DZN, and the specific site of the aptamer that strongly reacted with DZN was successfully identified using circular dichroism (CD) analysis. In presence of DZN, the aptamer and DZN formed a strong complex, thus providing an opportunity for hybridization of the DF probe and circular DNA, thereby initiating an RCA reaction. Repetitive poly thymine (T) sequence with a length of 30-mer, generated in the RCA reaction, served as a template for the synthesis of fluorescent copper nanoparticles, emitting an orange fluorescence signal (at approximately 620 nm) proportional to the amount of RCA product, within 10 min under UV irradiation. The CuNP fluorescence was imaged and quantified using an image analysis software. A linear correlation of the fluorescence signal was confirmed in the DZN concentration range of 0.1-3 ppm, with a detection limit of 0.15 ppm. Adoption of a label-free detection method, utilizing RCA and fluorescent CuNPs on COC substrates, reduced the need for complex equipment and requirements for DZN analysis, thereby representing a simple and rapid sensing method circumventing the limitations of current complex and labor-intensive methods. Electronic Supplementary Material ESM: The online version of this article (doi:10.1007/s12257-021-0220-0) contains supplementary material, which is available to authorized users.

Trends in sensor development toward next-generation point-of-care testing for mercury.

Mercury is one of the most common heavy metals and a major environmental pollutant that affects ecosystems. Since mercury and its compounds are toxic to humans, even at low concentrations, it is very important to monitor mercury contamination in water and foods. Although conventional mercury detection methods, including inductively coupled plasma mass spectrometry, atomic absorption spectroscopy, and gas chromatography-mass spectrometry, exhibit excellent sensitivity and accuracy, they require operation by an expert in a sophisticated and fully controlled laboratory environment. To overcome these limitations and realize point-of-care testing, many novel methods for direct sample analysis in the field have recently been developed by improving the speed and simplicity of detection. Commonly, these unconventional sensors rely on colorimetric, fluorescence, or electrochemical mechanisms to transduce signals from mercury. In the case of colorimetric and fluorescent sensors, benchtop methods have gradually evolved through technology convergence to give standalone platforms, such as paper-based assays and lab-on-a-chip systems, and portable measurement devices, such as smartphones. Electrochemical sensors that use screen-printed electrodes with carbon or metal nanomaterials or hybrid materials to improve sensitivity and stability also provide promising detection platforms. This review summarizes the current state of sensor platforms for the on-field detection of mercury with a focus on key features and recent developments. Furthermore, trends for next-generation mercury sensors are suggested based on a paradigm shift to the active integration of cutting-edge technologies, such as drones, systems based on artificial intelligence, machine learning, and three-dimensional printing, and high-quality smartphones.

3D-printed rolling circle amplification chip for on-site colorimetric detection of inorganic mercury in drinking water.

A point-of-care testing chip was developed for the colorimetric detection of inorganic mercury ion (Hg2+). The disposable chip fabricated by three-dimensional printing technology contains DNAzymes produced by rolling circle amplification (RCA); a color change caused by the enzymatic reaction between DNAzymes and the peroxidase substrate 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) is measured using a portable spectrophotometer. In the "turn-off"-type RCA reaction, the annealing of the T(12) primer that initiates the RCA reaction is blocked by the interaction of thymine with Hg2+; thus, the amount of amplified DNAzymes causing a color change is decreased depending on Hg2+ concentration. The colorimetric signal is enhanced by amplifying double-repeat DNAzymes from a circular DNA template. The chip detected Hg2+ in tap drinking water samples with high sensitivity (lowest validated value: 3.6 µg/L) and showed better selectivity, precision, and reproducibility than conventional analysis instruments. This low-cost easy-to-use platform can reduce the risk of accidental Hg2+ poisoning.

Efficient Production of Murine Uterine Damage Model.

BACKGROUND: Thin or damaged endometrium causes uterine factor-derived infertility resulting in a failure of embryonic implantation. Regeneration of endometrium is a major issue in gynecology and reproductive medicine. Various types of cells and scaffolds were studied to establish an effective therapeutic strategy. For this type of investigations, production of optimal animal models is indispensable. In this study, we tried to establish various murine uterine damage models and compared their features. METHODS: Three to ten-week-old C57BL/6 female mice were anesthetized using isoflurane. Chemical and mechanical methods using ethanol (EtOH) at 70 or 100% and copper scraper were compared to determine the most efficient condition. Damage of uterine tissue was induced either by vaginal or dorsal surgical approach. After 7-10 days, gross and microscopic morphology, safety and efficiency were compared among the groups. RESULTS: Both chemical and mechanical methods resulted in thinner endometrium and reduced number of glands. Gross morphology assessment revealed that the damaged regions of uteri showed various shapes including shrinkage or cystic dilatation of uterine horns. The duration of anesthesia significantly affected recovery after procedure. Uterine damage was most effectively induced by dorsal approach using 100% EtOH treatment compared to mechanical methods. CONCLUSION: Taken together, murine uterine damage models were most successfully established by chemical treatment. This production protocols could be applied further to larger animals such as non-human primate.

High-Throughput Screening of Acyl-CoA Thioesterase I Mutants Using a Fluid Array Platform.

Screening target microorganisms from a mutated recombinant library plays a crucial role in advancing synthetic biology and metabolic engineering. However, conventional screening tools have several limitations regarding throughput, cost, and labor. Here, we used the fluid array platform to conduct high-throughput screening (HTS) that identified Escherichia coli 'TesA thioesterase mutants producing elevated yields of free fatty acids (FFAs) from a large (106) mutant library. A growth-based screening method using a TetA-RFP fusion sensing mechanism and a reporter-based screening method using high-level FFA producing mutants were employed to identify these mutants via HTS. The platform was able to cover >95% of the mutation library, and it screened target cells from many arrays of the fluid array platform so that a post-analysis could be conducted by gas chromatography. The 'TesA mutation of each isolated mutant showing improved FFA production in E. coli was characterized, and its enhanced FFA production capability was confirmed.

Anti-melanogenesis effect of dehydroglyasperin C through the downregulation of MITF via the reduction of intracellular cAMP and acceleration of ERK activation in B16F1 melanoma cells.

BACKGROUND: In mammals, UV radiation induces melanin synthesis in melanocyte for protecting their skin through the stimulation of α-melanocyte stimulating hormone (α-MSH) from keratinocytes. In this study, the inhibitory effects of dehydroglyasperin C (DGC), an useful component of Glycyrrhiza uralensis (G. uralensis), was investigated on melanogenesis induced by α-melanocyte stimulating hormone (α-MSH) and its mechanisms. METHODS: Melanogenesis suppression effect of DGC on α-MSH induced B16F1 melanoma cells. The cell viability was measured by MTT assay. Expression and phosphorylation of melanogeic protein were conducted using western blot. cAMP acceleration was measured by cAMP immunoassay kit. To investigate whitening mechanism, we used ERK inhibitor (PD98059). RESULTS: DGC decreased intra cellular tyrosinase (TYR) activity and expression of melanin synthesis related proteins (TYR and TRP-1) in a dose-dependent manner on α-MSH induced melanogenesis. In addition, DGC induced the downregulation of MITF (melanocyte-specific transcription factor) through suppression of cAMP-CREB pathway. Also, phosphorylation of extracellular signal regulated kinase (ERK) decreased MITF by DGC treatment. CONCLUSION: Therefore, DGC could be used as a whitening ingredient in skin and clinical usage against hyperpigmentation.

A cracking-assisted micro-/nanofluidic fabrication platform for silver nanobelt arrays and nanosensors.

Nanowires (NWs) with a high surface-to-volume ratio are advantageous for bio- or chemical sensor applications with high sensitivity, high selectivity, rapid response, and low power consumption. However, NWs are typically fabricated by combining several nanofabrication and even microfabrication processes, resulting in drawbacks such as high fabrication cost, extensive labor, and long processing time. Here, we show a novel NW fabrication platform based on "crack-photolithography" to produce a micro-/nanofluidic channel network. Solutions were loaded along the microchannel, while chemical synthesis was performed in the nanoslit-like nanochannels for fabricating silver nanobelts (AgNBs). In addition, the NW/NB fabrication platform not only made it possible to produce AgNBs in a repeatable, high-throughput, and low-cost manner but also allowed the simultaneous synthesis and alignment of AgNBs on a chip, eliminating the need for special micro- and/or nanofabrication equipment and dramatically reducing the processing time, labor, and cost. Finally, we demonstrated that the AgNBs can be used as chemical sensors, either as prepared or when integrated in a flexible substrate, to detect target analytes such as hydrogen peroxide.

Nanoscale Hydrodynamic Film for Diffusive Mass Transport Control in Compartmentalized Microfluidic Chambers.

A compartmentalized microfluidic chamber array that offers not only separate cell culture environments but also independent control of the diffusion of small molecules provides an extremely useful platform for cell cultivations and versatile cellular assays. However, it is challenging to incorporate both cell compartmentalization and active diffusion control in real-time and precise manners. Here, we present a novel nanoscale hydrodynamic film (NHF) that is formed between a solid substrate and a polydimethylsiloxane (PDMS) surface. The thickness of the NHF can be adjusted by varying the pressure applied between them so that the mass transfer through the NHF can also be controlled. These novel phenomena are characterized and applied to develop a compartmentalized microchamber array with diffusion-tunable and solution-switchable chemostat-like versatile bacterial assays. The NHF-based compartmentalization technique is ideal for not only continuous bacterial cultivation by consistently refreshing various nutrient sources but also various diffusion-based microbial assays such as chemical induction of synthetically engineered bacterial cells and selective growth of a specific bacterial strain with respect to chemical environments. In addition, we show that tight compartmentalization protects cells in the chambers, while biofilm formation and nutrient contamination are eliminated by loading a lysis buffer, which typically hinders long-term continuous cultures and accurate microbial assays on a chip. Therefore, we ensure that the NHF-based compartmentalization platform proposed in this work will facilitate not only fundamental studies in microbiology but also various practical applications of microbes for production of valuable metabolites and byproducts in a high-throughput and highly efficient format.

A Microfluidic Platform for High-Throughput Screening of Small Mutant Libraries.

The screening and isolation of target microorganisms from mutated recombinant libraries are crucial for the advancement of synthetic biology and metabolic engineering. However, conventional screening tools present several limitations in throughput, cost, and labor. Herein, we describe a novel microfluidic high-throughput screening (HTS) platform with several advantages. The platform utilizes a fluid array to compartmentalize bacterial cells in well-ordered separated microwells and allows long-term cell culture with high throughput. The platform enables the extraction of selected target cells from the fluid array for additional culture and postanalysis by using a capillary-driven sample relocation method. To confirm the feasibility of the platform, we demonstrated two different types of HTS methods based on the levels of reporter gene expression and cellular growth rate difference. For the reporter gene-based HTS, a spike recovery approach was taken to demonstrate that target cells are successfully screened out from a mixture containing nontarget cells by repeating the culture and extraction processes. Additionally, the same platform allowed us to screen and sort target cells according to their cellular growth rate difference, which seems hard in conventional screening methods. Hence, the platform could be used for various microbiological assays, including the detection of cell-excreted metabolites, microbial biosensors, and other HTS systems.

Development of a highly specific and sensitive cadmium and lead microbial biosensor using synthetic CadC-T7 genetic circuitry.

Multiple copies of a cadC homolog encoding a heavy metal-responsive transcription factor were found in the genome of a bacterium isolated from ocean sediment, and the heavy metal responses of the encoded proteins were characterized using a fluorescence reporter assay. Each CadC regulator exhibited distinct specificity in response to heavy metal ions, indicating their potential use as modular heavy metal biosensors. Next, we constructed CadC-controlled T7 RNA transcription systems for intracellular signal amplification, i.e., higher sensitivity. Flow cytometry revealed that cadmium and lead ions could be recognized specifically by CadC-T7 biosensors, which could be combined with a microfluidic platform to generate heavy metal biosensor devices with increased sensitivity. Our results demonstrate the successful development of synthetic CadC-T7 genetic circuitry for use in improved heavy metal biosensor microfluidic devices.

Review of micro/nanotechnologies for microbial biosensors.

A microbial biosensor is an analytical device with a biologically integrated transducer that generates a measurable signal indicating the analyte concentration. This method is ideally suited for the analysis of extracellular chemicals and the environment, and for metabolic sensory regulation. Although microbial biosensors show promise for application in various detection fields, some limitations still remain such as poor selectivity, low sensitivity, and impractical portability. To overcome such limitations, microbial biosensors have been integrated with many recently developed micro/nanotechnologies and applied to a wide range of detection purposes. This review article discusses micro/nanotechnologies that have been integrated with microbial biosensors and summarizes recent advances and the applications achieved through such novel integration. Future perspectives on the combination of micro/nanotechnologies and microbial biosensors will be discussed, and the necessary developments and improvements will be strategically deliberated.

Cell penetrating peptide conjugated liposomes as transdermal delivery system of Polygonum aviculare L. extract.

In this study, Polygonum aviculare L. extract, which has superior antioxidative and cellular membrane protective activity, was loaded onto cell penetrating peptide (CPP) conjugated liposomes to enhance transdermal delivery. The physical characteristics of typical liposomes and CPP-conjugated liposomes containing P. aviculare extract were evaluated. The particle sizes of both liposomes were approximately 150 nm. Whereas the zeta potential of typical liposomes was -45 mV, that of CPP-conjugated liposomes was +42 mV. The loading efficiency of P. aviculare extract in both liposomes was calculated to be about 83%. Fluorescent-labeled liposomes were prepared to evaluate cellular uptake and skin permeation efficiency. Using flow cytometry, we found that CPP-conjugated liposomes improved cellular uptake of the fluorescent dye as compared with the typical liposomes. In addition, the skin permeation of CPP-conjugated liposomes was proved higher than that of typical liposomes by confocal laser scanning microscopy studies and Franz diffusion cell experiments. The improved cellular uptake and skin permeation of the CPP-conjugated liposomes were due to the cationic arginine-rich peptide. In vivo studies also determined that the CPP-conjugated liposomes were more effective in depigmentation and anti-wrinkle studies than typical liposomes. These results indicate that the CPP-conjugated liposomes could be effective for transdermal drug delivery of antioxidant and anti-aging therapeutics.

Chemostat-like microfluidic platform for highly sensitive detection of heavy metal ions using microbial biosensors.

Reporter-gene-based microbial biosensors have high potential for detecting small molecules, including heavy metal ions (HMIs), in a sensitive and selective manner by involving low costs. However, the sensitivity and dynamic range of the sensing mechanism are largely limited by the conventional culture environment that relies on the batch-type addition of the small molecules in nutrients and the subsequent genetic induction of sensing microbes. Here, we describe a high-throughput, chemostat-like microfluidic platform that can continuously supply both nutrients and inducers (HMIs) using microfabricated ratchet structures and a mixing microchannel network. We found that the microfluidic platform not only allowed microbial biosensors to be highly concentrated in a detection microchamber array but also enabled them to continuously grow and control synthetic genetic circuits in response to heavy metals. We also demonstrated that the combination of the platform and microbial biosensors enhanced the sensitivity for detecting divalent lead and cadmium ions by approximately three orders of magnitude relative to conventional batch-type methods. Because the platform is portable and only requires small sample volumes and fluorescent detection, the chemostat-like microfluidic platform in conjunction with microbial biosensors could be widely utilized to facilitate the specific and sensitive detection of molecular analytes on a chip.

Comparison of choroidal thickness in patients with diabetes by spectral-domain optical coherence tomography.

PURPOSE: To evaluate choroidal thickness in diabetes patients using spectral-domain optical coherence tomography. METHODS: We examined 203 eyes of 203 diabetic participants and 48 eyes of 48 healthy controls. The choroidal thickness at the foveal lesion was measured by enhanced-depth imaging optical coherence tomography. The participants were grouped according to diabetic retinopathy grade: no diabetic change, mild-to-moderate or severe non-proliferative, or proliferative diabetic retinopathy. The study parameters included history, age, axial length, intraocular pressure, central retinal thickness, fasting glucose, and blood pressure. RESULTS: The subfoveal choroidal thickness was thinner in eyes with non-proliferative or proliferative diabetic retinopathy than in normal eyes (p < 0.01). However, there was no difference between eyes with non-proliferative and proliferative diabetic retinopathy or between eyes with no diabetic change and the controls. Eyes exhibiting macular edema showed no significant difference in choroidal thickness compared with eyes having normal macular contours. CONCLUSIONS: The central choroid is thinner when eyes show diabetic changes on the retina. However, the presence of diabetic macular edema or proliferative change is not associated with more pronounced choroidal thinning.

Dry eye and tear film functions in patients with psoriasis.

PURPOSE: To evaluate dry eye symptoms, tear film function and ocular surface changes in patients with psoriasis. METHODS: The Dry Eye Questionnaire and ophthalmic examination including the Schirmer test, tear break-up time, corneal fluorescein test, meibomian gland obstruction and conjunctival impression cytology were assessed in patients with chronic plaque psoriasis. Results were compared between 30 patients and 30 healthy controls. RESULTS: The rate of positive responses in the Dry Eye Questionnaire and staining of corneal fluorescein test were significantly higher in the patients (P = 0.030) than in the controls (P = 0.012). The tear break-up time in patients was significantly lower than in the controls (P < 0.001). However, there were no differences in the Schirmer test and meibomian gland function between the patients and controls. In the impression cytology analysis, more cell alteration and decreased goblet cell density were observed in the patients (P < 0.001) compared with those obtained from controls (P = 0.003). CONCLUSIONS: The dry eye symptom was more common in patients with psoriasis. In addition, the patients showed a higher tear film instability and significant degeneration on the ocular surface when compared with the normal controls.

Comparison of intravitreal bevacizumab alone or combined with triamcinolone versus triamcinolone in diabetic macular edema: a randomized clinical trial.

PURPOSE: To compare the effect of an intravitreal injection of bevacizumab alone (IVB) or combined with triamcinolone (IVB/IVT) versus triamcinolone (IVT) in patients with diabetic macular edema (DME). METHODS: In this randomized three-arm clinical trial, eligible eyes were assigned randomly to one of the three study arms: the IVB group, 2 injections of 1.25 mg of bevacizumab with 6-week intervals; the IVB/IVT group, 1.25 mg of IVB with 2 mg of IVT, and the IVT group, 2 mg of IVT. The clinical course of best-corrected visual acuity and central macular thickness by optical coherence tomography was monitored for up to 12 months after the initial injection. RESULTS: One hundred eleven eyes of 105 patients with DME completed 12 months of follow-up. The IVB/IVT group and the IVT group showed better visual acuity and reduced central macular thickness at 6 weeks and 3 months, compared with the IVB group (p = 0.041, p = 0.02 at 6 weeks; p = 0.045, p = 0.043 at 3 months, respectively). However, no significant difference in visual acuity and central macular thickness was observed between the three groups at 12 months (p = 0.088, p = 0.132, respectively). The frequency of retreatment was lower in the IVB/IVT and IVT groups during the 12-month period (p < 0.001). No significant differences in visual acuity or central macular thickness were observed between the IVB/IVT and IVT groups during the follow-up. CONCLUSION: IVB/IVT and IVT showed more pronounced effects during the earlier postinjection period. However, levels of visual acuity or central macular thickness at 12 months were comparable in the three study groups. No beneficial effect of the combination injection was observed.

Surgical outcomes for primary rhegmatogenous retinal detachments in patients with pseudophakia after phacoemulsification.

PURPOSE: To evaluate the clinical features and surgical outcomes for primary rhegmatogenous retinal detachments (RDs) in patients with pseudophakia after phacoemulsification. METHODS: The medical records of patients with pseudophakia after phacoemulsification and intraocular lens implantation who had undergone surgery for primary rhegmatogenous RDs with a minimum duration of follow-up of 12 months were reviewed retrospectively. RESULTS: A total of 104 patients were enrolled in this study and 106 eyes were analyzed. Post-operative retinal attachment was achieved in 87 of the eyes (82.1%) and the final visual acuities (logarithm of the minimum angle of resolution) were improved to 0.65 ± 0.49 from the baseline measurement of 1.51 ± 1.14 (p < 0.001). Re-operations were performed in 24 of the eyes (22.6%) and there were no visible retinal breaks in 30 of the eyes (28.3%). The failure to identify a retinal break during surgery was associated with a lower rate of retinal reattachment, worse final visual acuity, and a higher rate of re-operation (p = 0.002, p = 0.02, and p = 0.002, respectively). The location of the identified retinal break was more common in the superotemporal quadrant than in the other quadrants. CONCLUSIONS: The inability to identify a retinal break during surgery was associated with a poor final outcome. Other factors were less important for the functional and anatomic success in patients with pseudophakic RDs.

Intravitreal bevacizumab (avastin) as an adjuvant for the treatment of posterior scleritis.

We report a case of posterior scleritis effectively managed with intravitreal bevacizumab. A 71-year-old woman was diagnosed with posterior scleritis. Although she was initially treated with systemic steroids, her clinical presentation deteriorated. She was then treated with a single intravitreal injection of bevacizumab and aqueous humor collection. The aqueous level of vascular endothelial growth factor prior to the intravitreal injection was 880.51 pg/mL, greater than that in the healthy control group (p < 0.001). One month later, the scleritis was completely resolved, and the patient remained stable during six months of follow-up. Intravitreal bevacizumab appears to be an effective adjuvant therapy for patients with posterior scleritis.