Reining in Cas13a activity with N-terminal removable tags expands Cas13a based molecular sensing and enables precise gene interference.

Activation of Cas13 is exclusively dependent on crRNA-target RNA hybridization according to the canonical mode of Cas13 action. Upon activation Cas13 can cleave both target RNA and any surrounding RNA. The latter has been well adopted by therapeutic gene interference and biosensor development. This work for the first time, rationale designs and validates a multi-component controlled activation system of Cas13 by N-terminus tagging. A composite SUMO tag comprised of His, Twinstrep, and Smt3 tags fully suppresses target dependent activation of Cas13a by interfering with crRNA docking. The suppression releases upon proteases mediated proteolytic cleavage. The modular composition of the composite tag can be altered to fulfill customized response to alternative proteases. The biosensor SUMO-Cas13a is able to resolve a broad concentration range of protease Ulp1 with a calculated LOD of 48.8pg/µL in aqueous buffer. Further, in accordance with this finding Cas13a was successfully programmed to exert target gene knock down preferentially in SUMO protease high cell types. In summary the discovered regulatory component not only fulfills Cas13a based protease detection for the first time, but also delivers a novel strategy for multi-component controlled activation of Cas13a toward temporal and spacial precision.

Lighting up ATP in cells and tissues using a simple aptamer-based fluorescent probe.

Extracellular ATP as a purinergic signaling molecule, together with ATP receptor, are playing an important role in tumor growth, therapy resistance, and host immunity suppression. Meanwhile ATP is a crucial indicator for cellular energy status and viability, thus a vital variable for tissue regeneration and in vitro tissue engineering. Most recent studies on COVID-19 virus suggest infection caused ATP deficit and release as a major characterization at the early stage of the disease and major causes for disease complications. Thus, imaging ATP molecule in both cellular and extracellular contexts has many applications in biology, engineering, and clinics. A sensitive and selective fluorescence "signal-on" probe for ATP detection was constructed, based on the base recognition between a black hole quencher (BHQ)-labeled aptamer oligonucleotide and a fluorophore (Cy5)-labeled reporter flare. The probe was able to detect ATP in solution with single digit µM detection limit. With the assistance of lipofectamine, this probe efficiently entered and shined in the model cells U2OS within 3 h. Further application of the probe in specific scenery, cardio-tissue engineering, was also tested where the ATP aptamer complex was able to sense cellular ATP status in a semi-quantitative manner, representing a novel approach for selection of functional cardiomyocytes for tissue engineering. At last a slight change in probe configuration in which a flexible intermolecular A14 linker was introduced granted regeneration capability. These data support the application of this probe in multiple circumstances where ATP measurement or imaging is on demand.

Using a glucose meter to quantitatively detect disease biomarkers through a universal nanozyme integrated lateral fluidic sensing platform.

Along with the advance in medical research, more biomarkers emerge as useful indicators for both disease and health index. However, majority of them have no practical or economic testing methods available yet, or rely on high-costing methods such as Enzyme-Linked Immuno-Sorbent Assay (ELISA), High-Performance Liquid Chromatography (HPLC), Mass Spectrum, and immunohistochemistry (IHC). In this article, we develop a universal nanozyme integrated testing platform for biological molecules that incorporates the electrochemical measurement of glucose with lateral flow immunostrip (LFS) for target analytes. This design involves the quantitative conversion of analytes into invertase and then glucose, which can be measured by an extremely affordable meter. The feasibility of this design was validated using 8-hydroxy-2'-deoxyguanosine (8-OHdG) and prostate specific antigen (PSA) as representatives for small molecules and moderate to large proteins respectively. Our approach yields results comparable to commercial diagnostic ELISA kits at a substantially reduced cost and reaction time. Specifically, the design has a detection limit of 0.23 ng mL-1 for 8-OHdG and 1.26 ng mL-1 for PSA, and a detection range of 0.1-100 ng mL-1 for 8-OHdG and 1-100 ng mL-1 for PSA. By combining the accessibility of well-established glucose testing and LFS, our design can serve as a point of care testing method that can be fully integrated into the personal lifestyle without requiring professional assistance.

Electrochemical Lateral Flow Paper Strip for Oxidative-Stress Induced DNA Damage Assessment.

The phrase "oxidative-stress induced DNA damage" is commonly used in both the scientific literature and common media outlets, and is frequently linked to detrimental elements of aging as well as the onset of illnesses. Due to the growing focus on this topic, a clear need has emerged to develop a quantitative, low-cost methodology to allow for periodic monitoring of oxidative-stress induced DNA damage within individuals. Recent literature examining the link between oxidative stress and the onset of various cancers has made monitoring an even more pressing need. The mechanism of oxidative-stress induced DNA damage originates in chronic inflammation, which in turn activates various transcription factors and diseases that influence the onset of tumor development, chemoresistance, radioresistance, and other harmful cellular processes. While current technologies that aim to provide quantitative metrics require extremely expensive equipment and significant technical expertise, our laboratory has designed a low-cost methodology utilizing a combination of carbon nanotubes, paper electrodes, and immunochromatographic strips.

Biosensing of DNA oxidative damage: a model of using glucose meter for non-glucose biomarker detection.

Non-glucose biomarker-DNA oxidative damage biomarker 8-hydroxy-2'-deoxyguanosine (8-OHdG) has been successfully detected using a smartphone-enabled glucose meter. Through a series of immune reactions and enzymatic reactions on a solid lateral flow platform, 8-OHdG concentration has been converted to a relative amount of glucose, and therefore can be detected by conventional glucose meter directly. The device was able to detect 8-OHdG concentrations in phosphate buffer saline as low as 1.73 ng mL-1 with a dynamic range of 1-200 ng mL-1. Considering the inherent advantages of the personal glucose meter, the demonstration of this device, therefore, should provide new opportunities for the monitoring of a wide range of biomarkers and various target analytes in connection with different molecular recognition events.

Microelectromechanical System-Based Sensing Arrays for Comparative in Vitro Nanotoxicity Assessment at Single Cell and Small Cell-Population Using Electrochemical Impedance Spectroscopy.

The traditional in vitro nanotoxicity assessment approaches are conducted on a monolayer of cell culture. However, to study a cell response without interference from the neighbor cells, a single cell study is necessary; especially in cases of neuronal, cancerous, and stem cells, wherein an individual cell's fate is often not explained by the whole cell population. Nonetheless, a single cell does not mimic the actual in vivo environment and lacks important information regarding cell communication with its microenvironment. Both a single cell and a cell population provide important and complementary information about cells' behaviors. In this research, we explored nanotoxicity assessment on a single cell and a small cell population using electrochemical impedance spectroscopy and a microelectromechanical system (MEMS) device. We demonstrated a controlled capture of PC12 cells in different-sized microwells (to capture a different number of cells) using a combined method of surface functionalization and dielectrophoresis. The present approach provides a rapid nanotoxicity response as compared to other conventional approaches. This is the first study, to our knowledge, which demonstrates a comparative response of a single cell and small cell colonies on the same MEMS platform, when exposed to metaloxide nanoparticles. We demonstrated that the microenvironment of a cell is also accountable for cells' behaviors and their responses to nanomaterials. The results of this experimental study open up a new hypothesis to be tested for identifying the role of cell communication in spreading toxicity in a cell population.

In situ amplification of intracellular microRNA with MNAzyme nanodevices for multiplexed imaging, logic operation, and controlled drug release.

MicroRNAs (miRNAs), as key regulators in gene expression networks, have participated in many biological processes, including cancer initiation, progression, and metastasis, indicative of potential diagnostic biomarkers and therapeutic targets. To tackle the low abundance of miRNAs in a single cell, we have developed programmable nanodevices with MNAzymes to realize stringent recognition and in situ amplification of intracellular miRNAs for multiplexed detection and controlled drug release. As a proof of concept, miR-21 and miR-145, respectively up- and down-expressed in most tumor tissues, were selected as endogenous cancer indicators and therapy triggers to test the efficacy of the photothermal nanodevices. The sequence programmability and specificity of MNAzyme motifs enabled the fluorescent turn-on probes not only to sensitively profile the distributions of miR-21/miR-145 in cell lysates of HeLa, HL-60, and NIH 3T3 (9632/0, 14147/0, 2047/421 copies per cell, respectively) but also to visualize trace amounts of miRNAs in a single cell, allowing logic operation for graded cancer risk assessment and dynamic monitoring of therapy response by confocal microscopy and flow cytometry. Furthermore, through general molecular design, the MNAzyme motifs could serve as three-dimensional gatekeepers to lock the doxorubicin inside the nanocarriers. The drug nanocarriers were exclusively internalized into the target tumor cells via aptamer-guided recognition and reopened by the endogenous miRNAs, where the drug release rates could be spatial-temporally controlled by the modulation of miRNA expression. Integrated with miRNA profiling techniques, the designed nanodevices can provide general strategy for disease diagnosis, prognosis, and combination treatment with chemotherapy and gene therapy.

[Analysis of mortality rate and causes of death among children under 5 years old in Beijing from 2003 to 2012].

OBJECTIVE: To understand the age-specific and cause-specific mortality rate among children under 5 years old in Beijing from 2003 to 2012. METHODS: Death surveillance data of children under the age of 5 were obtained from Beijing children mortality surveillance network from 2003 to 2012. Neonatal mortality rate (NMR), infant mortality rate (IMR), under 5-year old children mortality rate (U5MR) and the leading cause of death for under 5-year old children in urban, suburbs, and outer suburbs in Beijing were analyzed. RESULTS: The NMR, IMR and U5MR in Beijing were 2.08 (253/121 747), 3.11 (379/121 747) and 3.57 (435/121 747) per 1000 live births in 2012, respectively, which declined 54.88%, 50.24% and 54.75% compared with the level in 2003 respectively. The children mortality rates showed a decreasing trend in urban, suburb, and outer suburbs during 2003 and 2012 (NMR was decreased from 0.53%, 0.42%, and 0.48% in 2003 to 0.20%, 0.19%, and 0.23% in 2012; IMR was decreased from 0.73%, 0.58%, and 0.63% in 2003 to 0.30%, 0.29%, and 0.35% in 2012; U5MR was decreased from 0.90%, 0.72%, and 0.82% to 0.33%, 0.34%, and 0.39% in 2012, P < 0.01). There was a steady decline in the U5MR due to congenital heart disease, birth asphyxia, premature birth or low birth weight and traffic accident in Beijing from 2003 to 2012. The mortality rate of congenital heart disease declined from 140.63 to 41.89 per 100 000 live births, birth asphyxia declined from 109.38 to 59.96 per 100 000 live births, premature birth or low birth weight declined from 85.94 to 52.57 per 100 000 live births, traffic accident declined from 26.04 to 6.57 per 100 000 live births (P < 0.01). The mortality rate of congenital heart disease declined remarkably from 216.56 to 52.47, from 119.75 to 23.50, and from 134.58 to 63.11 per 100 000 live births in urban, suburb, and outer suburbs(P < 0.01). Six of the top 8 leading causes of death among children under 5 years old declined remarkably in rural areas. They were congenital heart disease, birth asphyxia, premature birth or low birth weight, traffic accident, drowning, and septicemia, and the mortality rate of them declined from 134.58 to 63.11, from 127.85 to 65.54, from 100.94 to 60.69, from 33.65 to 12.14, from 33.65 to 0.00, and from 26.92 to 4.85 per 100 000 live births, respectively (P < 0.05). There was no drowning death case in rural areas in recent 4 years. The top 5 leading causes of death among children under 5 years old in Beijing in 2012 were birth asphyxia, premature birth or low birth weight, congenital heart disease, pneumonia, and accidental suffocation. The mortality rate of these top 5 leading causes were 59.96, 52.57, 41.89, 24.64, and 15.61 per 100 000 live births in 2012. CONCLUSION: From 2003 to 2012, the NMR, IMR, U5MR and mortality rate of congenital heart disease declined remarkably in urban, suburb, and outer suburb areas in Beijing. There was a decrease trend for the six of the top 8 leading causes of death among children under 5 years old. The mortality rate of drowning dropped markedly in outer suburbs.

[Epidemiological study on the trend of accidental deaths among children under five in Beijing, during 2003-2012].

OBJECTIVE: To study the trend of accidental death among children under 5 years of age in Beijing. METHODS: Cases of death in children under 5 years old in Beijing, during 2003-2012 were collected, to analyze the strength and trends of accidental death, main causes of accident and its epidemiological features. RESULTS: The overall accidental death was 8.47% of all death among children under 5 years old in Beijing during 2003-2012. During these 10 years, data showed a downward trend on the mortality rates on injuries (P < 0.05), especially on drowning, in 1-4 year old and rural children under five years of age. In 2012, the accidental death rate of children under five was 6.37/105. The 5 main causes of accidental deaths were suffocation, traffic accident, falling, poisoning and drowning, in order of size. The main causes of death were suffocation in the 0-1 year group, suffocation and traffic accident in the 1-2 year group and traffic accident in the 3-4 year group. The proportion of deaths due to traffic accident increased gradually with age. Area distribution showed that accidental deaths mainly happened in rural area (52.19%), with two main types as traffic accident and suffocation. CONCLUSION: The reduction of accidental death rate among children under 5 years old in Beijing mainly was caused by the decline of accident mortality in 1-4 year old and children in the rural areas. Our data suggested that the focus in reducing the accidental death rate among children under 5 years old in Beijing should target on the prevention of infant suffocation and traffic accidents among the 1-4 year old, with rural areas in particular.

A paper electrode integrated lateral flow immunosensor for quantitative analysis of oxidative stress induced DNA damage.

A novel device combining electrochemical and colorimetric detection is developed for the rapid measurement of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a DNA oxidative damage biomarker. The device takes advantage of the speed and low cost of the conventional strip test as well as the high reliability and accuracy of the electrochemical assay. Competitive immunoreactions were performed on the lateral flow strip, and the captured 8-OHdG on the control line was determined by chronoamperometric measurement with carbon nanotube paper as the working electrode. At the same time, the color intensity of the test line was measured by a scanner and analyzed by the ImageJ software. The device was able to detect 8-OHdG concentrations in PBS as low as 2.07 ng mL(-1) by the colorimetric method and 3.11 ng mL(-1) by the electrochemical method. Furthermore, the device was successfully utilized to detect 8-OHdG in urine with a detection limit of 5.76 ng mL(-1) (colorimetric method) and 8.85 ng mL(-1) (electrochemical method), respectively. In conclusion, the integrated device with dual detection methods can provide a rapid, visual, quantitative and feasible detection method for 8-OHdG. The integration of these two methods holds two major advantages over tests based on a single method. Firstly, it can provide double confidence on the same assay. Secondly, by involving two methods that differ in principle, the integration could potentially avoid false results coming from one method. In addition, these methods do not require expensive equipment or trained personnel, making it suitable for use as a simple, economical, portable field kit for on-site monitoring of 8-OHdG in a variety of clinical settings.

Chip based single cell analysis for nanotoxicity assessment.

Nanomaterials, because of their tunable properties and performances, have been utilized extensively in everyday life related consumable products and technology. On exposure, beyond the physiological range, nanomaterials cause health risks via affecting the function of organisms, genomic systems, and even the central nervous system. Thus, new analytical approaches for nanotoxicity assessment to verify the feasibility of nanomaterials for future use are in demand. The conventional analytical techniques, such as spectrophotometric assay-based techniques, usually require a lengthy and time-consuming process and often produce false positives, and often cannot be implemented at a single cell level measurement for studying cell behavior without interference from its surrounding environment. Hence, there is a demand for a precise, accurate, sensitive assessment for toxicity using single cells. Recently, due to the advantages of automation of fluids and minimization of human errors, the integration of a cell-on-a-chip (CoC) with a microfluidic system is in practice for nanotoxicity assessments. This review explains nanotoxicity and its assessment approaches with advantages/limitations and new approaches to overcome the confines of traditional techniques. Recent advances in nanotoxicity assessment using a CoC integrated with a microfluidic system are also discussed in this review, which may be of use for nanotoxicity assessment and diagnostics.

Lab-on-chip device for single cell trapping and analysis.

Traditional cell assay gives us an average result of multiple cells and it is assumed that the resultant is the outcome of all cells in population. However, single cell studies have revealed that individual cells of same type may differ dramatically and these differences may have important role to play in cells functionality. Such information can be obscured in only studying cell population experimental approach. To uncover biological principles and ultimately to improve the detection and treatment of disease, new approaches are highly required to single cell analysis. We propose to fabricate a lab on chip device to study high throughput single cell nanotoxicity analysis. The chip incorporates independently addressable active microwell electrodes for cell manipulation and analysis. We employed positive-dielectrophoresis approach to quickly and efficiently capture single cells in each wells with having control over individual microwells. We examined change in impedance properties to verify cell capture in microwell and its health and present a novel model of single cell assay for nanotoxicity, and drug testing.

Biosensing approaches for rapid genotoxicity and cytotoxicity assays upon nanomaterial exposure.

The increased utilization of nanomaterials could affect human health and the environment due to increased exposure. Several mechanisms regarding the negative effects of nanomaterials have been proposed, one of the most discussed being oxidative stress. Many studies have shown that some metal oxide nanoparticles can enhance reactive oxygen species generation, inducing oxidative stress, DNA damage, and unregulated cell signaling, and eventually leading to changes in cell motility, apoptosis, and even carcinogenesis. 8-Hydroxy-2'-deoxyguanosine (8-OHdG) is one of the predominant forms of oxidative DNA damage, and has therefore been widely used as a biomarker for oxidative stress and carcinogenesis. Ther are two major objectives to this study. Firstly, the development of a novel lateral flow immunoassay (LFIA) is presented to measure the concentration of 8-OHdG in cells and thus reveal the nanotoxicity on the genomic level. The feasibility of this new method is validated by comparison with two other established methods: Alamar Blue assay and a recently developed electrical impedance sensing (EIS) system on the level of cell proliferation/viability. Secondly, the toxicological effects of three metallic nanoparticles (CuO, CdO, and TiO2 ) are investigated and compared using these three methods with completely different mechanisms. The results show that there is a high variation among different nanoparticles concerning their ability to cause toxic effects. CuO nanoparticles are the most potent regarding cytotoxicity and DNA damage. CdO shows a fallen cell viability as well as DNA damage, however, to a lesser extent than CuO nanoparticles. TiO2 particles only cause very limited cytotoxicity, and there is no obvious increase in 8-OHdG levels. In conclusion, LFIA as well as the EIS system are useful methods for quantitative or qualitative nanotoxicity assessments with high sensitivity, specificity, speed of performance, and simplicity.

Development of paper-based analytical kit for point-of-care testing.

Paper-based analytical devices have been widely used for biomedical, environmental and food-quality testing. This review focuses on paper-based tests for biomarkers and bacterial detection with a brief introduction about various fabrication techniques and designs, biological and nonbiological probes and detection methods. Paper is relatively cheap and available in abundance. Moreover, properties of paper such as it being disposable, easy to use and store, and that it is easy to transport and modify draw significant attention to it as a platform for the development of paper-based analytical devices. These traits make paper-based analytical devices a strong candidate in point-of-care devices for rapid and economical testing near the site of patients.

Paper based point-of-care testing disc for multiplex whole cell bacteria analysis.

Point-of-care testing (POCT) of infectious bacterial agents offers substantial benefits for disease diagnosis, mainly by shortening the time required to obtain results and by making the test available bedside or at remote care centers. Immunochromatographic lateral flow biosensors offer a low cost, highly sensitive platform for POCT. In this article, we describe the fabrication and testing of a multiplex immuno-disc sensor for the specific detection of Pseudomonas aeruginosa and Staphylococcus aureus. Antibody conjugated gold nanoparticles were used as the signaling agents. The detection range of the bacteria lies within 500-5000 CFU/ml. The advantage of the immuno-disc sensor is that it does not require any preprocessing of biological sample and is capable of whole cell bacterial detection. We also describe the design and fabrication of a compact portable device which converts the color intensity of the gold nanoparticles that accumulate at the test region into a quantitative voltage reading proportional to the bacterial concentration in the sample. The combination of the immuno-disc and the portable color reader provides a rapid, sensitive, low cost, and quantitative tool for the detection of a panel of infectious agents present in the patient sample.