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Abstract With both foodborne illness and food spoilage detrimentally impacting human health and the economy, there is growing interest in the development of in situ sensors that offer real-time monitoring of food quality within enclosed food packages. While oligonucleotide-based fluorescent sensors have illustrated significant promise, the development of such on-food sensors requires consideration towards sensing-relevant fluorescence properties of target food products—information that has not yet been reported. To address this need, comprehensive fluorescence profiles for various contamination-prone food products are established in this study across several wavelengths and timepoints. The intensity of these food backgrounds is further contextualized to biomolecule-mediated sensing using overlaid fluorescent oligonucleotide arrays, which offer perspective towards the viability of distinct wavelengths and fluorophores for in situ food monitoring. Results show that biosensing in the Cyanine3 range is optimal for all tested foods, with the Cyanine5 range offering comparable performance with meat products specifically. Moreover, recognizing that mass fabrication of on-food sensors requires rapid and simple deposition of sensing agents onto packaging substrates, RNA-cleaving fluorescent nucleic acid probes are successfully deposited via microcontact printing for the first time. Direct incorporation onto food packaging yields cost-effective sensors with performance comparable to ones produced using conventional deposition strategies.

Deep-shaft mining in roadway stress concentration, difficult control of the surrounding rock, and mining imbalance affect mining efficiency in coal mines. The mechanical parameters and crack development law of roadway surrounding rock were studied by laboratory experiments. The mechanical model was established by the method of theoretical analysis, the maximum empty roof distance was deduced, and two support schemes were designed. Through the numerical simulation method, the support scheme was compared and analyzed, the deformation rule of roadway surrounding rock, the optimization of roadway support parameters and the application of hysteresis technology are studied. The effect of different support schemes was verified by three-dimensional similar simulation experiments. The practical engineering verification showed that the construction time of each support circle was reduced by approximately 1 h, and the work was completed 60 days earlier. The research showed that the optimal support scheme was support scheme 1 (7 bolts with 800 mm × 1000 mm spacing between the roof, 10 bolts with 800 mm × 1000 mm spacing between the two sides, and 2 × 1 × 2 cables with 1600 mm × 2000 mm spacing between the roof). Support scheme 1 was applied to the engineering site to control the deformation of surrounding rock at 80 mm, and the deformation was less than the original support scheme. The construction was completed in advance under the hysteresis process, and the support efficiency and operation safety were improved. The results revealed the mechanism of surrounding rock control and proved the effectiveness of digging support synergy. This optimization plan serves as a reference for studying roadway support in rapid excavation and provides theoretical support for safe and efficient coal roadway mining.

A growing number of viruses pose significant threats to human health, impact agricultural production and compromise food safety. Minimizing the impact of viral infections demands for effective technologies that can deliver sensitive viral detection under various settings. Functional nucleic acids (FNAs) – natural or synthetic nucleic acid molecules with catalytic and/or binding properties – have been increasingly explored as key components for setting up diagnostic tests for viral detection. FNA-based viral biosensors offer distinct advantages, such as the ability to achieve sensitive viral detection in complex biological samples, ease of use, scalability, and cost-effectiveness. This mini-review will be dedicated to appraising progress made in recent years in FNA-based biosensors for diagnosing diverse human and animal viruses. We will first describe virus structures and potential biomarkers that can be utilized as the targets for FNA probe engineering. This is followed by reviewing the latest advances in two classes of FNAs - DNAzyme and DNA aptamers, with focuses on DNAzyme- and DNA aptamer-based biosensors that employ diverse signal transduction mechanisms to achieve sensitive viral detections. Finally, we will discuss current challenges in the field and suggest future research directions that can address these challenges.

Abstract There is a need for point‐of‐care bacterial sensing and identification technologies that are rapid and simple to operate. Technologies that do not rely on growth cultures, nucleic acid amplification, step‐wise reagent addition, and complex sample processing are the key for meeting this need. Herein, multiple materials technologies are integrated for overcoming the obstacles in creating rapid and one‐pot bacterial sensing platforms. Liquid‐infused nanoelectrodes are developed for reducing nonspecific binding on the transducer surface; bacterium‐specific RNA‐cleaving DNAzymes are used for bacterial identification; and redox DNA barcodes embedded into DNAzymes are used for binding‐induced electrochemical signal transduction. The resultant single‐step and one‐pot assay demonstrates a limit‐of‐detection of 102 CFU mL−1, with high specificity in identifying Escherichia coli amongst other Gram positive and negative bacteria including Klebsiella pneumoniae, Staphylococcus aureus, and Bacillus subtilis. Additionally, this assay is evaluated for analyzing 31 clinically obtained urine samples, demonstrating a clinical sensitivity of 100% and specify of 100%. When challenging this assay with nine clinical blood cultures, E. coli‐positive and E. coli‐negative samples can be distinguished with a probability of p

Abstract Saliva is an attractive sample for coronavirus disease 2019 testing due its ease of collection and amenability to detect viral RNA with minimal processing. Using a direct-to-RT-PCR method with saliva self-collected from confirmed COVID-19 positive volunteers, we observed 32% false negative results. Confirmed negative and healthy volunteer samples spiked with 106 genome copies/mL of heat-inactivated severe acute respiratory syndrome coronavirus 2 showed false negative results of 10% and 13%, respectively. Additional sample heating or dilution of the false negative samples conferred only modest improvements. These results highlight the potential to significantly underdiagnose COVID-19 infections when testing directly from minimally processed heterogeneous saliva samples.

Displaying extremely high peroxidase-like activity and uniform cubic structure enclosed by (100) facets, Pd-Ir nanocubes are an attractive nanomaterial for bioanalysis. However, there exists a great challenge to deposit atomic layers of Ir on the surface of Pd nanocubes due to the relatively low energy barrier of homogeneous nucleation of Ir atoms compared to heterogeneous nucleation. Here, a simple and surfactant-free approach is presented to synthesize Pd-Ir nanocubes with atomic Ir shell thickness in an aqueous solution at room temperature. Biomolecules such as antibodies and nucleic acids have free access to the surface of Pd-Ir nanocubes. Applications of Pd-Ir nanocubes in immunoassays and aptamer-based biosensors are realized, exploiting the excellent peroxidase activity and fluorescence quenching ability of Pd-Ir nanocubes. This work makes a significant step forward towards the practical utility of Pd-Ir nanocubes in bioanalysis.

The mechanical model of the basic roof fracture structure is established on the basis of key block theory to study the roof breaking mechanism of gob-side entry retaining under roof cutting and pressure relief, and the analytical formula of roof support resistance is derived when the key block of the basic roof is stable. The influence of roof cutting angle and cutting height on roof support resistance is also analyzed. Determining the cutting seam parameters of the retained roadway roof is necessary to identify the support resistance of the roadway roof due to the correlation between the roof cutting parameters and the support resistance. Taking the II 632 haulage drift of the Hengyuan coal mine as the engineering background, FLAC3D numerical simulation is used in this paper to analyze the influence of different roof cutting angles and cutting heights on the surrounding rock structure evolution of retained roadways. Results show that the roof cutting angle and cutting height respond to the support resistance of the retained roadway roof, and the support resistance required by the roof increases with the roof cutting angle and cutting height. This condition ensures that the side roof of the gob can be cut off smoothly, and the support resistance required by the roof of retained roadways is within a reasonable range. Through theoretical and numerical simulation analysis, the reasonable roof cutting height of II 632 haulage drift is 8 m and the roof cutting angle is 15°. The theoretical analysis and numerical simulation results reveal that the required support resistance to maintain the stability of the roadway roof is 0.38 MPa. The supporting scheme of the roof of the II 632 haulage drift in the Hengyuan coal mine is then designed. Finally, the field industrial test is used for verification. The borehole imaging results show that the overall line of the retained roadway roof is small based on the description of field monitoring results. The deformation of the surrounding rock surface of the retained roadway is less than 100 mm, and the roadway is 40 m from the lagging working face. The deformation rate of surrounding rock decreases with the increase in distance from the working face. The integrity of the retained roadway roof is good, and the deformation of the surrounding rock is effectively controlled.

Due to their relative synthetic and chemical simplicity compared to antibodies, aptamers afford enhanced stability and functionality for the detection of environmental contaminants and for use in environmental monitoring. Furthermore, nucleic acid aptamers can be selected for toxic targets which may prove difficult for antibody development. Of particular relevance, aptamers have been selected and used to develop biosensors for environmental contaminants such as heavy metals, small-molecule agricultural toxins, and water-borne bacterial pathogens. This review will focus on recent aptamer-based developments for the detection of diverse environmental contaminants. Within this domain, aptamers have been combined with other technologies to develop biosensors with various signal outputs. The goal of much of this work is to develop cost-effective, user-friendly detection methods that can complement or replace traditional environmental monitoring strategies. This review will highlight recent examples in this area. Additionally, with innovative developments such as wearable devices, sentinel materials, and lab-on-a-chip designs, there exists significant potential for the development of multifunctional aptamer-based biosensors for environmental monitoring. Examples of these technologies will also be highlighted. Finally, a critical perspective on the field, and thoughts on future research directions will be offered.

The application of roof-cutting and pressure-relief gob-side entry retention plays a critical role in controlling the stability of the surrounding rock at the entry, easing continuity tension and improving resource recovery ratio. The excavation of the 360,803 airway in Xinji No. 1 Mine is affected by intense mining of the 360,805 working face. Hence, to address the stability problem of surrounding rock in the 360,803 airway, rock mass blast weakening theory was used in this study to analyze the blasting stress of columnar charged rock mass and obtain the radiuses of crushed, fractured, and vibration zones under uncoupled charging conditions. The reasonable array pitch, length, and dip angle of boreholes were determined according to the pressure-relief range of the blasting fracture. The migration laws of roof strata were explored based on a mechanical model of overlying roof strata structure on the working face. Subsequently, the horizon, breaking span, and caving sequence of hard roof strata were obtained to determine the roof-cutting height of this entry. On the basis of the theory of key stratum, the number of sequences at the roof caving limit stratum and hanging roof length in the goaf were calculated, the analytical solution to critical coal pillar width was acquired, the evaluation indexes for the stability of entry-protecting coal pillars were determined, and the engineering requirements for the 25 m entry-protecting coal pillars in the 360,803 airway were met. Moreover, various indexes such as roof separation fracture, displacement of surrounding rock, and loose circle of surrounding rock in the gob-side entry were analyzed. The stability and cementation status of surrounding rock in the 360,803 airway were evaluated, and tunneling safety was ensured.

Abstract We report on a new colorimetric DNA detection method that takes advantage of the power of polymerase chain reaction (PCR) and the simplicity of the classic litmus test. The strategy makes use of a modified set of primers for PCR to facilitate ensuing manipulations of resultant DNA amplicons: their tagging with urease and immobilization onto magnetic beads. The amplicon/urease-laden beads are then used to hydrolyze urea, resulting in the increase of pH that can be conveniently reported by a pH-sensitive dye. We have successfully applied this strategy for the detection of two hypervirulent strains of the bacterium Clostridium difficile that are responsible for the recent increase in the global incidence and severity of C. difficile infections. Furthermore, the viability of this test for diagnostic applications is demonstrated using clinically validated stool samples from C. difficile infected patients.

DNA nanostructures with interlocked topologies will tend to display different behaviour to the linear counterparts. Here, the authors show a DNA catenane that is inactive for rolling circle amplification but is activated upon cleavage of one ring, and exploit this for the development of a biosensing system.

Ingestion of water containing toxic contaminants above levels deemed safe for human consumption can occur unknowingly since numerous common contaminants in drinking water are colorless and odorless. Uranyl is particularly problematic as it has been found at dangerous levels in sources of drinking water. Detection of this heavy metal-ion species in drinking water currently requires sending a sample to a laboratory where trained personnel use equipment to perform the analysis and turn-around times can be long. A pH-responsive colorimetric biosensor was developed to enable detection of uranyl in water which coupled the uranyl-specific 39E DNAzyme as a recognition element, and an enzyme capable of producing a pH change as the reporter element. The rapid colorimetric assay presented herein can detect uranyl in lake and well water at concentrations relevant for environmental monitoring, as demonstrated by the detection of uranyl at levels below the limits set for drinking water by major regulatory agencies including the World Health Organization (30 μg/L). This simple and inexpensive DNAzyme-based assay enabled equipment-free visual detection of 15 μg/L uranyl, using both solution-based and paper-based pH-dependent visualization strategies.

The elucidation of the cellular processes involved in vitamin and cofactor biosynthesis is a challenging task. The conventional approaches to these investigations rely on the discovery and purification of the products (i.e proteins and metabolites) of a particular transport or biosynthetic pathway, prior to their subsequent analysis. However, the purification of low-abundance proteins or metabolites is a formidable undertaking that presents considerable technical challenges. As a solution, we present an alternative approach to such studies that circumvents the purification step. The proposed approach takes advantage of: (1) the molecular detection capabilities of a riboswitch-based sensor to detect the cellular levels of its cognate molecule, as a means to probe the integrity of the transport and biosynthetic pathways of the target molecule in cells, (2) the high-throughput screening ability of fluorescence-activated cell sorters to isolate cells in which only these specific pathways are disrupted, and (3) the ability of next-generation sequencing to quickly identify the genes of the FACS-sorted populations. This approach was named "RiboFACSeq". Following their identification by RiboFACSeq, the role of these genes in the presumed pathway needs to be verified through appropriate functional assays. To demonstrate the utility of our approach, an adenosylcobalamin (AdoCbl)-responsive riboswitch-based sensor was used in this study to demonstrate that RiboFACSeq can be used to track and sort cells carrying genetic mutations in known AdoCbl transport and biosynthesis genes with desirable sensitivity and specificity. This method could potentially be used to elucidate any pathway of interest, as long as a suitable riboswitch-based sensor can be created. We believe that RiboFACSeq would be especially useful for the elucidation of biological pathways in which the proteins and/or their metabolites are present at very low physiological concentrations in cells, as is the case with vitamin and cofactor biosynthesis.

Bacterial detection plays an important role in protecting public health and safety, and thus, substantial research efforts have been directed at developing bacterial sensing methods that are sensitive, specific, inexpensive, and easy to use. We have recently reported a novel “mix-and-read” assay where a fluorogenic DNAzyme probe was used to detect model bacterium E. coli. In this work, we carried out a series of optimization experiments in order to improve the performance of this assay. The optimized assay can achieve a detection limit of 1000 colony-forming units (CFU) without a culturing step and is able to detect 1 CFU following as short as 4 h of bacterial culturing in a growth medium. Overall, our effort has led to the development of a highly sensitive and easy-to-use fluorescent bacterial detection assay that employs a catalytic DNA.

When not constrained to long double-helical arrangements, DNA is capable of forming structural arrangements that enable specific sequences to perform functions such as binding and catalysis under defined conditions. Through a process called in vitro selection, numerous catalytic DNAs, known as deoxyribozymes or DNAzymes, have been isolated. Many of these molecules have the potential to act as therapeutic agents and diagnostic tools. As such, a better understanding of the structural arrangements present in these functional DNAs will aid further efforts in the development and optimization of these useful molecules. Structural characterization of several deoxyribozymes through mutagenesis, in vitro re-selection, chemical probing and circular dichroism has revealed many distinct and elaborate structural classes. Deoxyribozymes have been found to contain diverse structural elements including helical junctions, pseudoknots, triplexes, and guanine quadruplexes. Some of these studies have further shown the repeated isolation of similar structural motifs in independent selection experiments for the same type of chemical reaction, suggesting that some structural motifs are well suited for catalyzing a specific chemical reaction. To investigate the extent of structural diversity possible in deoxyribozymes, a group of kinase deoxyribozymes have been extensively characterized. Such studies have discovered some interesting structural features of these DNAzymes while revealing some novel DNA structures.

As the key constituents of the genetic code, the importance of nucleic acids to life has long been appreciated. Despite being composed of only four structurally similar nucleotides, single-stranded nucleic acids, as in single-stranded DNAs and RNAs, can fold into distinct three-dimensional shapes due to specific intramolecular interactions and carry out functions beyond serving as templates for protein synthesis. These functional nucleic acids (FNAs) can catalyze chemical reactions, regulate gene expression, and recognize target molecules. Aptamers, whose name is derived from the Latin word aptus meaning “to fitâ€Â, are oligonucleotides that can bind their target ligands with high affinity and specificity. Since aptamers exist in nature but can also be artificially isolated from pools of random nucleic acids through a process called in vitro selection, they can potentially bind a diverse array of compounds. In this review, we will discuss the research that is being done to develop aptamers against various biomolecules, the progress in engineering biosensors by coupling aptamers to signal transducers, and the prospect of employing these sensors for a range of chemical and biological applications. Advances in aptamer technology emphasizes that nucleic acids are not only the fundamental molecules of life, they can also serve as research tools to enhance our understanding of life. The possibility of using aptamer-based tools in drug discovery and the identification of infectious agents can ultimately augment our quality of life.

Recently, in vitro studies have demonstrated that adiponectin has antiangiogenic and tumor growth-limiting properties. Additionally, serum adiponectin levels have been associated with the risk of several cancers; specifically, serum adiponectin was significantly lower in lung cancer patients with advanced-stage disease. In this study, we examined the association of adiponectin gene promoter variations associated with adiponectin gene expression and plasma levels in non-small cell lung cancer (NSCLC) in a Han Chinese population. A total of 319 patients with NSCLC and 489 healthy individuals were recruited to evaluate the association of four adiponectin gene promoter single-nucleotide polymorphisms (SNPs) (SNP-12140G>A, SNP-11426A>G, SNP-11391G>A and SNP-11377C>G) with NSCLS risk. Additionally, we constructed haplotypes of these four SNPs and evaluated the association of these haplotypes with NSCLS risk. Our results showed that among these four SNPs, only SNP-12140G>A was associated with NSCLC risk (P0.05). Additionally, an association analysis of the four SNPs stratified into pathologic stages I+II and III+IV showed that these SNPs did not exhibit significant differences between pathologic stages I+II and III+IV. Moreover, we did not observe any differences in allele and genotype frequency for these SNPs between adenocarcinoma and squamous cell carcinoma. Our results indicated that the G allele of SNP-12140 may be a risk factor for NSCLC (OR = 1.516; 95% CI: 1.098-2.094) in this Han Chinese population.

Many DNAzymes have been isolated from synthetic DNA pools to cleave natural RNA (D-RNA) substrates and some have been utilized for the design of aptazyme biosensors for bioanalytical applications. Even though these biosensors perform well in simple sample matrices, they do not function effectively in complex biological samples due to ubiquitous RNases that can efficiently cleave D-RNA substrates. To overcome this issue, we set out to develop DNAzymes that cleave L-RNA, the enantiomer of D-RNA, which is known to be completely resistant to RNases. Through in vitro selection we isolated three L-RNA-cleaving DNAzymes from a random-sequence DNA pool. The most active DNAzyme exhibits a catalytic rate constant ~3 min-1 and has a structure that contains a kissing loop, a structural motif that has never been observed with D-RNA-cleaving DNAzymes. Furthermore we have used this DNAzyme and a well-known ATP-binding DNA aptamer to construct an aptazyme sensor and demonstrated that this biosensor can achieve ATP detection in biological samples that contain RNases. The current work lays the foundation for exploring RNA-cleaving DNAzymes for engineering biosensors that are compatible with complex biological samples.

Pathogenic bacteria such as Escherichia coli O157, Salmonella and Campylobacter are the main causes for food and waterborne illnesses. Lysis of these bacteria is an important component of the sample preparation for molecular identification of these pathogens. The pathogenicity of these bacteria is so high that they cause illness at very low concentrations (1–10 CFU/100 mL). Hence, there is a need to develop methods to collect a small number of such bacterial cells from a large sample volume and process them in an automated reagent-free manner. An electrical method to concentrate the bacteria and lyse them has been chosen here as it is reagent free and hence more conducive for online and automated sample preparation. We use commercially available nanoporous membranes sandwiched between two microfluidic channels to create thousands of parallel nanopore traps for bacteria, electrophoretically accumulate and then lyse them. The nanopores produce a high local electric field for lysis at moderate applied voltages, which could simplify instrumentation and enables lysis of the bacteria as it approaches them under an appropriate range of electric field (>1000 V/cm). Accumulation and lysis of bacteria on the nanoporous membrane is demonstrated by using the LIVE/DEAD BacLight Bacterial Viability Kit and quantified by fluorescence intensity measurements. The efficiency of the device was determined through bacterial culture of the lysate and was found to be 90% when a potential of 300 V was applied for 3 min.

Geniposide is a key iridoid glycoside from Gardenia jasminoides fructus widely used in traditional Chinese herbal medicine. However, detection of this small molecule represents a significant challenge mostly due to the lack of specific molecular recognition elements. In this study, we have performed in vitro selection experiments to isolate DNA aptamers that can specifically bind geniposide. Using a stringent selection procedure, we have isolated DNA aptamers that can distinguish geniposide from genipin and glucose, two structural analogs of geniposide. Two top aptamers exhibit low micromolar binding affinity towards geniposide, but show significantly reduced affinity to genipin and glucose. These aptamers have the potential to be further developed into analytical tools for the detection of geniposide.

Determination of accurate dosage of existing antibiotics and discovery of new antimicrobials or probiotics entail simple but effective methods that can conveniently track bacteria growth and inhibition. Here we explore the application of a previously reported fluorogenic E. coli-specific DNAzyme (catalytic DNA), RFD-EC1, as a molecular probe for monitoring bacterial inhibition exerted by antibiotics and for studying bacterial competition as a result of cohabitation. Because the DNAzyme method provides a convenient way to monitor the growth of E. coli, it is capable of determining the minimal inhibitory concentration (MIC) of antibiotics much faster than the conventional optical density (OD) method. In addition, since the target for RFD-EC1 is an extracellular protein molecule from E. coli, RFD-EC1 is able to identify pore-forming antibiotics or compounds that can cause membrane leakage. Finally, RFD-EC1 can be used to analyse the competition of cohabitating bacteria, specifically the inhibition of growth of E. coli by Bacillus subtilis. The current work represents the first exploration of a catalytic DNA for microbiological applications and showcases the utility of bacteria-sensing fluorogenic DNAzymes as simple molecular probes to facilitate antibiotic and probiotic research.

Biosensors are analytical devices that have found a variety of applications in medical diagnostics, food quality control, environmental monitoring and biodefense. In recent years, functional nucleic acids, such as aptamers and nucleic acid enzymes, have shown great potential in biosensor development due to their excellent ability in target recognition and catalysis. Deoxyribozymes (or DNAzymes) are single-stranded DNA molecules with catalytic activity and can be isolated to recognize a wide range of analytes through the process of in vitro selection. By using various signal transduction mechanisms, DNAzymes can be engineered into fluorescent, colorimetric, electrochemical and chemiluminescent biosensors. Among them, colorimetric sensors represent an attractive option as the signal can be easily detected by the naked eye. This reduces reliance on complex and expensive equipment. In this review, we will discuss the recent progress in the development of colorimetric biosensors that make use of DNAzymes and the prospect of employing these sensors in a range of chemical and biological applications.

The process of in vitro selection has led to the discovery of many aptamers with potential to be developed into inhibitors and biosensors, but problems in isolating aptamers against certain targets with desired affinity and specificity still remain. One possible improvement is to use libraries enhanced for motifs repeatedly isolated in aptamer molecules. One such frequently observed motif is the two-tiered guanine quadruplex. In this study we investigated whether DNA libraries could be designed to contain a large fraction of molecules capable of folding into two-tiered guanine quadruplexes. Using comprehensive circular dichroism analysis, we found that DNA libraries could be designed to contain a large proportion of sequences that adopt guanine quadruplex structures. Analysis of individual sequences from a small library revealed a mixture of quadruplexes of different topologies providing the diversity desired for an in vitro selection. We also found that primer-binding sites are detrimental to quadruplex formation and devised a method for post-selection amplification of primer-less quadruplex libraries. With the development of guanine quadruplex enriched DNA libraries, it should be possible to improve the chances of isolating aptamers that utilize a quadruplex scaffold and enhance the success of in vitro selection experiments.

The development of the in vitro selection technique has allowed the isolation of functional nucleic acids, including catalytic DNA molecules (DNAzymes), from random-sequence pools. The first-ever catalytic DNA obtained by this technique in 1994 is a DNAzyme that cleaves RNA. Since then, many other RNase-like DNAzymes have been reported from multiple in vitro selection studies. The discovery of various RNase DNAzymes has in turn stimulated the exploration of these enzymatic species for innovative applications in many different areas of research, including therapeutics, biosensing, and DNA nanotechnology. One particular research topic that has received considerable attention for the past decade is the development of RNase DNAzymes into fluorescent reporters for biosensing applications. This paper provides a concise survey of the most significant achievements within this research topic.

Most NAE (nucleic acid enzyme) sensors are composed of an RNA-cleaving catalytic motif and an aptameric receptor. They operate by activating or repressing the catalytic activity of a relevant NAE through the conformational change in the aptamer upon target binding. To transduce a molecular recognition event to a fluorescence signal, a fluorophore-quencher pair is attached to opposite ends of the RNA substrate such that when the NAE cleaves the substrate, an increased level of fluorescence can be generated. However, almost all NAE sensors to date harbor either NAEs that cannot accommodate a fluorophore-quencher pair near the cleavage site or those that can accept such a modification but require divalent transition metal ions for catalysis. Therefore, the signaling magnitude and the versatility of current NAE sensors might not suffice for analytical and biological applications. Here we report an RNA-cleaving DNA enzyme, termed 'MgZ', which depends on Mg(2+) for its activity and can accommodate bulky dye moieties next to the cleavage site. MgZ was created by in vitro selection. The selection scheme entailed acidic buffering and ethanol-based reaction stoppage to remove selfish DNAs. Characterization of MgZ revealed a three-way junction structure, a cleavage rate of 1 min(-1), and 26-fold fluorescence enhancement. Two ligand-responsive NAE sensors were rationally designed by linking an aptamer sequence to the substrate of MgZ. In the absence of the target, the aptamer-linked substrate is locked into a conformation that prohibits MgZ from accessing the substrate. In the presence of the target, the aptamer releases the substrate, which induces MgZ-mediated RNA cleavage. The discovery of MgZ and the introduction of the above NAE sensor design strategy should facilitate future efforts in sensor engineering.

The Gejiu region in southeast Yunnan Province was an important metal and ore production area in China as early as the Han Dynasty (202 B.C.-A.D. 220). Past archaeological investigations in this region discovered an ancient smelting site. Large numbers of smelting-related relics including lead discs, copper ingots, and smelting wastes were also excavated from the Heimajing cemetery site nearby. These discoveries suggest that the burial occupants were probably involved in ancient mining, metal smelting, and the metal production industry. An archaeometallurgical analysis of smelting-related relics was conducted. The results demonstrated that lead, copper, and different copper-base alloys were produced in the region. The chemical compositional analysis and lead isotope studies of the samples collected from the Heimajing cemetery site suggest that there was probably a production and supply system and a demand for copper-base products in the Dian Kingdom (now central Yunnan) and Jiaozhi Commandery (now northern Vietnam) through cross-regional resource allocation. The Gejiu region therefore might have had a complete industry chain on the southwest frontier of the Han Empire. KEYWORDS: Han Empire, Heimajing cemetery, intermediate products, archaeometallurgy, lead isotope., INTRODUCTION The early metal production centers of mainland Southeast Asia and in the Yunnan Province of China, along with the circulation and exchange networks for early intermediate metal and copper-base [...]