A label-free aptasensing method for detecting SARS-CoV-2 virus antigen by using dumbbell probe-mediated circle-to-circle amplification.

Controlling the spread of pathogen requires an efficient and accurate diagnosis. Compared with nucleic acid and antibody detection, antigen assays are more convenient to meet clinical diagnostic needs. However, antigen detection is often difficult to achieve high sensitivity in a limited time. In this work, a novel aptasensing method was designed for the purpose of SARS-CoV-2 antigen detection, using a dumbbell padlock probe-mediated circle-to-circle amplification (C2CA) approach. A sandwich complex of antibody-antigen-aptamer is first formed on the magnetic beads. Afterwards, the signal is amplified by a C2CA reaction involving two tandem rolling circle amplifications. Without special instruments or nanomaterials, a detection limit of 575 fg/mL for S1 protein can be achieved in less than 2 h. In the case of the spike pseudovirus SARS-CoV-2 in artificial saliva, the detection limit is 272 TU/µL, which is much lower than average viral load in patients. Therefore, our method provides a timely, efficient and accurate approach for the clinical diagnosis of SARS-CoV-2. It also opens up the application of C2CA in aptamer sensing and antigen detection.

A novel label-free capillary electrophoresis LED-induced fluorescence platform based on catalytic hairpin assembly for sensitive detection of multiple circulating tumor DNA.

Circulating tumor DNA (ctDNA) is a highly promising biomarker for the early diagnosis and treatment of gastric cancer (GC). However, there is still a lack of effective and practical ctDNA detection methods. In this work, a simple and economical capillary non-gel sieving electrophoresis-LED induced fluorescence detection (NGCE-LEDIF) platform coupled with catalytic hairpin assembly (CHA) as the signal amplification strategy is proposed for quantitative detection of PIK3CA E542K and TP53 (two types of ctDNA associated with GC). We have reasonably designed two pairs of programmable oligonucleotide hairpin probes for PIK3CA E542K and TP53. Using a one-pot reaction, the presence of ctDNA triggers the cyclic amplification of CHA, forming numerous thermodynamically stable H1/H2 double-strands. The H1/H2 double-stranded DNA catalyzed by PIK3CA E542K and TP53 can be easily separated by NGCE due to their different lengths, enabling simultaneous detection of both ctDNAs. Under optimal experimental conditions, the detection limits of this strategy for detecting GC-related biomarkers PIK3CA E542K and TP53 are 20.35 pM and 19.61 pM, respectively, and can achieve 730-fold signal amplification. This strategy has a good recovery in the serum matrix. The results of this study show that this strategy has significant advantages such as high selectivity, a simple process, no special instruments and equipment, no need for fluorescence modification of hairpin probes in advance, high automation, low cost, and minimal sample consumption. This provides a powerful method for the detection of trace cancer biomarkers in the serum matrix with good application prospects.

In-Depth Serum Proteomics Reveals the Trajectory of Hallmarks of Cancer in Hepatitis B Virus-Related Liver Diseases.

Hepatocellular carcinoma (HCC) is a prevalent cancer in China, with chronic hepatitis B (CHB) and liver cirrhosis (LC) being high-risk factors for developing HCC. Here, we determined the serum proteomes (762 proteins) of 125 healthy controls and Hepatitis B virus-infected CHB, LC, and HCC patients and constructed the first cancerous trajectory of liver diseases. The results not only reveal that the majority of altered biological processes were involved in the hallmarks of cancer (inflammation, metastasis, metabolism, vasculature, and coagulation) but also identify potential therapeutic targets in cancerous pathways (i.e., IL17 signaling pathway). Notably, the biomarker panels for detecting HCC in CHB and LC high-risk populations were further developed using machine learning in two cohorts comprised of 200 samples (discovery cohort = 125 and validation cohort = 75). The protein signatures significantly improved the area under the receiver operating characteristic curve of HCC (CHB discovery and validation cohort = 0.953 and 0.891, respectively; LC discovery and validation cohort = 0.966 and 0.818, respectively) compared to using the traditional biomarker, alpha-fetoprotein, alone. Finally, selected biomarkers were validated with parallel reaction monitoring mass spectrometry in an additional cohort (n = 120). Altogether, our results provide fundamental insights into the continuous changes of cancer biology processes in liver diseases and identify candidate protein targets for early detection and intervention.

An ultrasensitive fluorescence aptasensor for SARS-CoV-2 antigen based on hyperbranched rolling circle amplification.

Sensitive and accurate diagnosis of SARS-CoV-2 infection at early stages can help to attenuate the effects of the COVID-19. Compared to RNA and antibodies detection, direct detection of viral antigens could reflect infectivity more appropriately. However, it is still a great challenge to construct a convenient, accurate and sensitive biosensor with a suitable molecular recognition element for SARS-CoV-2 antigens. Herein, we report a HRCA-based aptasensor for simple, ultrasensitive and quantitative detection of SARS-CoV-2 S1 protein and pseudovirus. The aptamer sequence used here is selected from several published aptamers by enzyme-linked oligonucleotide assay and molecular docking simulation. The sensor forms an antibody-target-aptamer sandwich complex on the surface of microplates and elicits HRCA for fluorescent detection. Without complicated operations or special instruments and reagents, the aptasensor can detect S1 protein with a LOD of 89.7 fg/mL in the linear range of 100 fg/mL to 1 µg/mL. And it can also detect SARS-CoV-2 spike pseudovirus in artificial saliva with a LOD of 51 TU/µL. Therefore, this simple and ultrasensitive aptasensor has the potential to detect SARS-CoV-2 infection at early stages. It may improve the timeliness and accuracy of SARS-CoV-2 diagnosis and demonstrate a strategy to conduct aptasensors for other targets.

Web hybrid chain reaction enhanced fluorescent magnetic bead array for digital nucleic acid detection.

The detection of biomarkers at low concentrations is important in clinical diagnostic analyses and has attracted continuous research. In this work, absolute quantification of hepatitis B virus (HBV) DNA was achieved using magnetic beads with isothermal, enzyme-free DNA nanostructure for fluorescence amplification. Firstly, the DNA-functionalized bead captured the target nucleic acid in the form of sandwich hybridization, and the individual target lighted up the entire bead by isothermal web hybridization chain reaction (wHCR). After the microarray scanning, the target nucleic acids can be digitally quantified based on the Poisson statistics. Therefore, the fluorescent bead assay enabled precise detection of HBV DNA down to 5 fM level without external calibration curves. Moreover, this method not only specifically distinguished single-base mismatched sequences, but also obtained the quantitative detection of HBV DNA in serum samples. Unlike routine digital detection usually combined with complex compartment partitioning operations, the amplification structure immobilized on beads can be conducted in microcentrifuge tubes with a volume of microliter scale. This work expands the application of magnetic beads in the digital quantitative detection via enzyme-free and isothermal method.

Highly-sensitive and simple fluorescent aptasensor for 17 b-estradiol detection coupled with HCR-HRP structure.

As an important kind of environmental endocrine disruptors, 17 ß -Estradiol (E2) plays a major role in affecting the growth of human including sexual characters, pregnancy system, etc. In the modern society, with the threat of abuse in breeding, it is imperative to design sensitive methods for detecting low concentration of E2 in environment. In this work, we constructed a highly sensitive and simple fluorescent aptasenor for detecting E2 via amplification of hybridization chain reaction (HCR) and horseradish peroxidase (HRP). Through the competitions between complementary strand (cmDNA) and E2 to E2 aptamer modified on magnetic beads, the unbound cmDNA would be collected and captured by polystyrene microspheres to induce HCR which brought abundant biotin sites. Subsequently, benefit from the excellent catalytic performance of streptavidin-horseradish peroxidase (SA-HRP), the highly sensitive fluorescence signals could be obtained in low concentration of E2. Under the optimal conditions, the prospered method for E2 detection was shown a good liner range from 1 to 100 pg/mL, with the lower detecting limit of 0.2 pg/mL compared with previous work. In addition, the recovery rates tested in the real samples of milk and water were 99.20%-108.06% and 91.07%-106.13%. In all, the assay may provide a perspective way for highly sensitive detection for various contaminants in the real samples.

Fully Automatic Multi-Class Multi-Residue Analysis of Veterinary Drugs Simultaneously in an Integrated Chip-MS Platform.

Microfluidic chip analysis has great potential advantages such as high integration, fast speed analysis, and automatic operation and is widely used not only in biological fields but also in many other analytical areas such as agriculture and food safety. Herein, a fully automatic multi-class multi-residue analysis of veterinary drugs simultaneously in an integrated chip-mass spectrometry (chip-MS) platform was developed. The developed microfluidic chip platform integrated three modules including the extraction and filtration module, "pass-through" clean-up module, and online evaporation module. The resulting chip has been coupled to a MS detector successfully, in which 23 kinds of residues in five classes were simultaneously qualitatively and quantitatively detected without chromatographic separation, obtaining the limits of detection of the spiked milk sample in the range of 0.23-4.13 ng/mL and the recovery rate in the range from 71.7 to 118.0% under optimized conditions. The microfluidic chip system developed in this study provided a new idea for the development of detection chips and exhibited considerable potential in the point-of-care testing in milk.

A Cascaded DNA Circuit in Bead Arrays for Quantitative Single-Cell MicroRNA Analysis.

Single-cell microRNA (miRNA) analysis helps people understand the causes of diseases and formulate new disease treatment strategies. However, miRNA from a single cell is usually very rare and requires signal amplification for accurate quantification. Here, to amplify the signal, we constructed the cascaded DNA circuits consisting of catalytic hairpin assembly and hybrid chain reaction into the bead array platform, on which the uniformly distributed beads were adopted for miRNA quantification. After exponential signal amplification, a consistent linear correlation between the percentage of fluorescent beads and the copy number of miRNA was detected. The proposed bead array can achieve ultrahigh sensitivity as low as 60 copies of miR-155 and high specificity for distinguishing single nucleotide differences. This method has been successfully applied to the quantitative detection of miRNA in a single cancer cell. The high sensitivity, programmability, and simple workflow of the bead array chip will give a huge advantage in basic and clinical research.

MoS2-Assisted LDI Mass Spectrometry for the Detection of Small Molecules and Quantitative Analysis of Sulfonamides in Serum.

A two-dimensional MoS2 nanosheet was prepared by a chemical exfoliation method and served as an excellent matrix for the detection of small molecules by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). In comparison with organic matrices (CHCA, 3-AQ) and a graphene matrix, we found that a MoS2 matrix showed better performance in analysis of amino acids, peptides, fatty acids, and sulfonamides. A systematic comparison of the MoS2 matrix with both ion modes showed that mass spectra produced in negative ion mode featured a corresponding single deprotonated ion, which was rather different from the complex multiple alkali metal addition peaks present in positive ion mode. In addition, better sensitivity and reproducibility were obtained in negative ion mode. The ionization mechanism of MoS2 as a matrix in negative ion mode was further discussed. The deproton peak intensity of the analyte fatty acid decreased after the addition of the hole-scavenger KSCN, indicating that the ionization of the fatty acid was caused by the Auger complex effect of MoS2 and electron injection. Experiments have shown that the MoS2 matrix detects small molecules with good repeatability and can perform semiquantitative analysis of sulfonamides. Finally, the MoS2 matrix was employed for quantitative determination of sulfamethoxine in serum samples by an internal standard method. This MoS2-assisted laser desorption/ionization mass spectrometry (MoS2-assisted LDI MS) method provides a simple, rapid, high-throughput approach to evaluate the drug levels in the patient serum and can achieve convenient drug therapy monitoring.

A fluorescent microarray platform based on catalytic hairpin assembly for MicroRNAs detection.

The DNA microarray has distinctive advantages of high-throughput and less complicated operations, but tends to have a relatively low sensitivity. Catalytic hairpin assembly (CHA) is one of the most promising enzyme-free, isothermal DNA circuit for high efficient signal amplification. Here, a microarray-based catalytic hairpin assembly (mi-CHA) biosensing method has been developed to detect various miRNAs in a single test simultaneously. The target miRNA can trigger conformational transformations of hairpin-structured DNA probes on the chip surface and lead to the specific signal amplification. A significant advantage of this approach is that each duplex produced by the solid-phase CHA will be immobilized on the certain location of the chip and release fluorescent signal via the universal domain, eliminating the requirement of different fluorophores. This method has manifested a high detection sensitivity of human cancer-associated miRNAs (miR-21 and miR-155) down to 1.33 fM and promised a high specificity to distinguish single-base mismatches. Furthermore, the practicability of this method was demonstrated by analyzing target miRNAs in human serum and cancer cells. The experimental results suggest that the proposed method has high-throughput analytical potential and could be applied to many other clinical diagnosis.

Accomplishment of one-step specific PCR and evaluated SELEX process by a dual-microfluidic amplified system.

One of the main obstacles for systematic evolution of ligands by exponential enrichment (SELEX) failure is the generation of a non-specific product, as selection-inherent amplification procedures tend to form by-products, which prevents the enrichment of target-binding aptamers. Herein, we reported a dual-microfluidic amplified system (dual-MAS) based on the real-time polymerase chain reaction (PCR) detection chip and the large volume PCR chip for one-step specific PCR and for evaluating the SELEX process. First, it is a simple method to accomplish analytical PCR and amplification PCR in one step, and the optimal number of cycles for generating the specific PCR product is the cycles when the slope of the linear amplification period of the real-time PCR curve begins to decrease. Second, the time used by the dual-MAS for generating a specific PCR product is reduced to 30 min, and the multi-functional dual-MAS can simultaneously evaluate the SELEX process by providing important information on the amounts of enriched sequences and the library diversity in every round of SELEX. In addition, pollution contamination and fragment loss can be significantly avoided in the closed chip. Last, the specific PCR product, the amounts of enriched sequences, and the library diversity can be obtained for every single SELEX in just 30 min. Compared with current methods, this system can reduce the time for generating a specific PCR product and SELEX, and it is easier to choose the optimal number of cycles for a specific PCR product. In a word, it is a sensitive, simple, and rapid strategy to improve the specificity of the PCR product and make the process of SELEX in a controlled way.

Screening aptamers for serine ß-lactamase-expressing bacteria with Precision-SELEX.

Klebsiella pneumoniae carbapenemase 2 (KPC-2) is a serine ß-lactamase that can hydrolyze almost all ß-lactam antibiotics. The drug resistant problem of bacteria expressing carbapenemases is currently a global problem, therefore, rapid and specific detection of pathogenic bacteria is urgent. In order to obtain an aptamer that can specifically recognize bacteria expressing KPC-2, we have established a method called Precision-SELEX. Precision-SELEX combined protein SELEX and bacterium SELEX. In this method, KPC-2 was used as a target protein, and Escherichia coli expressing KPC-2 (KPC-2 E. coli) was used as a target bacterium. After precision-SELEX, the same aptamer named XK-10 that can recognize KPC-2 and KPC-2 E. coli was obtained while the screening process could be shortened to 4 rounds. Dissociation equilibrium constants were calculated as 0.81 nM by SPR. In addition, XK-10 could specifically bind to KPC-2 E. coli, which was confirmed through flow cytometry and molecular Docking Simulations. The high-content imaging method could detect KPC-2 E. coli. In all, the Precision-SELEX provides an accurate and efficient method to screening aptamers for bacteria.

On-line multi-residue analysis of fluoroquinolones and amantadine based on an integrated microfluidic chip coupled to triple quadrupole mass spectrometry.

An on-line multi-residue qualitative and quantitative analysis method for fluoroquinolones and amantadine using an integrated microfluidic chip was developed prior to directly coupling to triple quadrupole mass spectrometry (QQQ-MS). Six parallel channels consisting of sample filtration units and micro solid phase extraction (micro-SPE) columns were present in the specifically designed microfluidic device. Firstly, the impurities in the sample solution were trapped by the micropillars in the filtration units. The solution passed through the micro-SPE units packed with hydrophilic-lipophilic balanced (HLB) particles, and then the two classes of drugs were enriched. After washing, the targets were eluted and immediately electrosprayed for MS analysis. This approach allowed effective filtration, enrichment, elution, and MS detection without the introduction of an additional separation step after SPE. Direct electrospray ionization (ESI)-MS in multiple reaction monitoring (MRM) mode could not only ensure the high sensitivity of quantitative analysis, but also achieved accurate qualitative analysis towards targets using the MRM ratios, reducing the possibility of false positives. Good linear relationships were obtained by the internal standard (IS) method with a linear range of 1-200 ng mL-1 (R2 > 0.992). The mean recoveries of the eight target analytes were from 85.2% to 122% with the relative standard deviation (RSD) ranging from 5.6% to 20.3%. All this demonstrated that the developed microfluidic device could be a useful tool for rapid detection in the field of food safety.

A novel method combining aptamer-Ag10NPs based microfluidic biochip with bright field imaging for detection of KPC-2-expressing bacteria.

The ß-lactam drugs resistance poses a serious threat to human health throughout the world. Klebsiella pneumoniae carbapenemase 2 (KPC-2) is a carbapenemase that produced in bacteria can hydrolyze carbapenems, which typically considered as the antibiotics of last resort. Therefore, there is an urgent need to quickly and accurately detect whether bacteria express KPC-2. In this paper, a PDMS/glass microfluidic biochip integrated with aptamer-modified Ag10NPs nano-biosensors was developed for rapid, simple and specific pathogenic bacteria detection, more importantly, the biochip was combined with bright field imaging, then the captured bacteria could be observed and counted directly without using extra chemical labeling. KPC-2-expressing Escherichia coli (KPC-2 E.coli) was used as the target bacterium with a detected limit of 102 CFU and capture efficiency exceeded 90%. This method is remarkably specific towards KPC-2 E.coli over other non-resistant bacteria, and pathogen assay only takes ∼1 h to complete in a ready-to-use microfluidic biochip. Furthermore, the effective capture and fast counting of microfluidic biochip system demonstrates its potential for the rapid detection of antibiotic-resistant bacteria.

The discovery of lactoferrin dual aptamers through surface plasmon resonance imaging combined with a bioinformation analysis.

An analytical method for screening aptamers for different recognition sites in lactoferrin (Lac) molecules has been developed based on Surface Plasmon Resonance imaging (SPRi), combined with the cluster classification calculation of a quasi-aptamer library strategy and molecular docking simulation analysis. Using the software simulation, a homology analysis was performed on the selected quasi-aptamer sequences, which could be divided into 8 different families. Based on the principle of biomolecular recognition, a label-free, high-throughput dual immune site screening method was established, in which the nucleic acid aptamers of recognizing ability for lactoferrin molecules were fixed onto the surface of the SPRi sensor chip and could bind to the lactoferrin molecules. Then, the aptamer candidates to be paired were introduced, and the recognition event of the second immune site was judged by observing the binding signal of SPRi. The paired SPRi signal was generated only when the site identified by the second nucleic acid molecule was different from the first immune site. Based on this principle, a pair of Lac nucleic acid aptamers (Lac-8 and Lac-25) was finally screened and confirmed using computerized simulation, and has been employed to assay Lac in milk by ELONA (Enzyme-Linked Oligonucleotide Assay).

COVID-19 diagnostic testing: Technology perspective.

The corona virus disease 2019 (COVID-19) is a highly contagious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). More than 18 million people were infected with a total of 0.7 million deaths in ∼188 countries. Controlling the spread of SARS-CoV-2 is therefore inherently dependent on identifying and isolating infected individuals, especially since COVID-19 can result in little to no symptoms. Here, we provide a comprehensive review of the different primary technologies used to test for COVID-19 infection, discuss the advantages and disadvantages of each technology, and highlight the studies that have employed them. We also describe technologies that have the potential to accelerate SARS-CoV-2 detection in the future, including digital PCR, CRISPR, and microarray. Finally, remaining challenges in COVID-19 diagnostic testing are discussed, including (a) the lack of universal standards for diagnostic testing; (b) the identification of appropriate sample collection site(s); (c) the difficulty in performing large population screening; and (d) the limited understanding of SARS-COV-2 viral invasion, replication, and transmission.

Highly Integrated Microfluidic Chip Coupled to Mass Spectrometry for Online Analysis of Residual Quinolones in Milk.

In this work, a highly integrated microfluidic chip with multifunction coupled to mass spectrometry (MS) was developed for online analysis of seven different regulated quinolones (QNs) in milk samples. Procedures of sample extraction, immunoaffinity enrichment, magnetic separation, and online elution were performed simultaneously on the specifically designed device. Based on the specificity of antibodies, direct (electrospray ionization) ESI-MS at full scan mode without liquid chromatography (LC) separation and further tandem mass spectrometry (MS/MS) analysis was developed for the identification of target QNs. One single isotope internal standard (IS) method was presented for quantitative analysis of seven QNs. Upon targeted online extraction and enrichment by antibody conjugated magnetic beads, seven QNs were quantitatively determined by the IS method with the linear range of 0.2/0.5-10 ng/mL (R2 > 0.991). The limits of detection (LODs) for the seven QNs were in the range of 0.047-0.490 ng/mL. This system permits automated on-chip immunoaffinity enrichment and accurate MS detection without additional off-line cleanup procedures.

Enzyme-free fluorescence microarray for determination of hepatitis B virus DNA based on silver nanoparticle aggregates-assisted signal amplification.

In this study, we designed a fluorescence enhancement strategy based on silver nanoparticle (AgNP) aggregates for the detection of hepatitis B virus DNA sequences. AgNPs were functioned with recognition probes (Cy3-probe) and hybrid probes (Oligomer-A and Oligomer-B). The presence of target DNA mediated the formation of sandwich complexes between the immobilized capture probes and the functionalized AgNPs, which was followed by hybridization-induced formation of AgNP aggregates. The fluorescent intensity could be extremely amplified by both the increasing number of fluorophores and metal enhanced fluorescence (MEF) effect. Under optimal conditions, this method achieved a detection limit of 50 fM which was 1560-fold lower than that of un-enhanced fluorescent assays. It was illustrated that the HBV DNA concentrations ranging from 100 fM to 10 nM had a good log-linear correlation with the corresponding fluorescent intensity (R = 0.991). Moreover, this method had high specificity both for distinguishing single-base mismatches and identifying target DNA under the interference of genomic DNA. This fluorescent microarray had high-throughput analytical potential and could apply to many other disease diagnoses.

In-depth serum proteomics reveals biomarkers of psoriasis severity and response to traditional Chinese medicine.

Serum and plasma contain abundant biological information that reflect the body's physiological and pathological conditions and are therefore a valuable sample type for disease biomarkers. However, comprehensive profiling of the serological proteome is challenging due to the wide range of protein concentrations in serum. Methods: To address this challenge, we developed a novel in-depth serum proteomics platform capable of analyzing the serum proteome across ~10 orders or magnitude by combining data obtained from Data Independent Acquisition Mass Spectrometry (DIA-MS) and customizable antibody microarrays. Results: Using psoriasis as a proof-of-concept disease model, we screened 50 serum proteomes from healthy controls and psoriasis patients before and after treatment with traditional Chinese medicine (YinXieLing) on our in-depth serum proteomics platform. We identified 106 differentially-expressed proteins in psoriasis patients involved in psoriasis-relevant biological processes, such as blood coagulation, inflammation, apoptosis and angiogenesis signaling pathways. In addition, unbiased clustering and principle component analysis revealed 58 proteins discriminating healthy volunteers from psoriasis patients and 12 proteins distinguishing responders from non-responders to YinXieLing. To further demonstrate the clinical utility of our platform, we performed correlation analyses between serum proteomes and psoriasis activity and found a positive association between the psoriasis area and severity index (PASI) score with three serum proteins (PI3, CCL22, IL-12B). Conclusion: Taken together, these results demonstrate the clinical utility of our in-depth serum proteomics platform to identify specific diagnostic and predictive biomarkers of psoriasis and other immune-mediated diseases.

High-density micro-well array with aptamer-silver conjugates for cell sorting and imaging at single cells.

Characterizing cell behavior is important to modern medical diagnoses as the changes of cell behavior are often indicators of huge diseases. In order to gain enough information about cells, developing novel methods of cell sorting and imaging is an important task. With development of micro-fabrication technologies, more advanced miniaturized devices are applied to cell research. Here, a portable and easy-to-use chip with high-density periodic micro-well array is designed and fabricated to capture target cells specifically. Combining with aptamer-silver conjugates and FAM functioned report probes, the sandwich assay was successfully applied for imaging cells. Any well of the chip is carefully designed to provide abundant information on single cells. Since there are 19,200 microwells in a single chip, more information is available. Compared to other cells, such as HEK-293, MCF-7, U2OS and Ramos cells, the sandwich assay shows high specificity towards target cell CCRF-CEM. What's more, the applications of the chip can be further expanded to other cells imaging if suitable aptamers were selected. This high-density micro-well array of aptamer-silver conjugates is hopeful to play an important role in medical diagnosis in the future.