Background-suppressed tumor-targeted photoacoustic imaging using bacterial carriers.

Photoacoustic (PA) imaging offers promise for biomedical applications due to its ability to image deep within biological tissues while providing detailed molecular information; however, its detection sensitivity is limited by high background signals that arise from endogenous chromophores. Genetic reporter proteins with photoswitchable properties enable the removal of background signals through the subtraction of PA images for each light-absorbing form. Unfortunately, the application of photoswitchable chromoproteins for tumor-targeted imaging has been hampered by the lack of an effective targeted delivery scheme; that is, photoswitchable probes must be delivered in vivo with high targeting efficiency and specificity. To overcome this limitation, we have developed a tumor-targeting delivery system in which tumor-homing bacteria (Escherichia coli) are exploited as carriers to affect the point-specific delivery of genetically encoded photochromic probes to the tumor area. To improve the efficiency of the desired background suppression, we engineered a phytochrome-based reporter protein (mDrBphP-PCMm/F469W) that displays higher photoswitching contrast than those in the current state of the art. Photoacoustic computed tomography was applied to achieve good depth and resolution in the context of in vivo (mice) imaging. The present system effectively integrates a genetically encoded phytochrome-based reporter protein, PA imaging, and synthetic biology (GPS), to achieve essentially background-suppressed tumor-targeted PA monitoring in deep-seated tissues. The ability to image tumors at substantial depths may enable target-specific cancer diagnoses to be made with greater sensitivity, fidelity, and specificity.

A Photoelectrochemical Nanoreactor for Single-Cell Sampling and Near Zero-Background Faradaic Detection of Intracellular microRNA.

Rational utilization of the rich light-bio-matter interplay taking place in single-cell analysis represents a new technological direction in the field. The light-fueled operation is expected to achieve advanced photoelectrochemical (PEC) single-cell analysis with unknown possibilities. Here, a PEC nanoreactor capable of single-cell sampling and near zero-background Faradaic detection of intracellular microRNA (miR) is devised by the construction of a small reaction chamber accommodating the target-triggered hybridization chain reaction for binding the metallointercalator of [Ru(bpy)2 (dppz)]2+ as the signal reporter. Light stimulation of the dsDNA/metallointercalator adduct will induce the generation of photocurrents, underpinning a zero-biased and near zero-background PEC method toward Faradaic detection of non-electrogenic miR at the single-cell level. Using this nanotool, lower miR concentration in the near-nucleus region than that in the main cytosol was revealed.

Ultrasensitive zero-background photoelectrochemical biosensor for analysis of organophosphorus pesticide based on in situ formation of DNA-templated Ag2S photoactive materials.

Herein, a novel signal-on photoelectrochemical (PEC) biosensor with nearly zero background noise (ZBN) was first fabricated to determine the presence of organophosphorus pesticide based on in situ formation of DNA-templated Ag2S photoactive materials, accompanied by hybridization chain reaction (HCR) signal amplification. The capture probe (S1) on the gold nanoparticle-modified electrode can hybridize with the aptamer molecule to generate a simple PEC biosensor. In the presence of a target molecule, the aptamer molecule is released on the double-stranded DNA (dsDNA)-modified PEC biosensor. Meanwhile, the capture probe remains on the electrode and can open the DNA hairpins (H1, H2) which are rich in cytosine, to trigger the HCR reaction. The rich "C" strands are uncovered after formation of a long dsDNA polymer strand, which can assemble multiple silver ions (Ag+) by means of by C-Ag+-C chelation. Then, a large number of Ag2S can be generated by challenging with S2- solution, producing a satisfactory photocurrent signal. The photoactive material is formed in situ, which eliminates the laborious operation. Moreover, the signal can be highly amplified with nearly zero background noise and HCR signal amplification. Under optimal conditions, the ZBN aptasensor exhibited high sensitivity and selectivity, with a low detection limit of 2 pg mL-1 for malathion. Importantly, the sensing platform can also be applied to determine the presence of malathion in real samples. In this assay, a novel signal-on photoelectrochemical biosensor with nearly zero background noise was first fabricated to determine the presence of organophosphorus pesticide based on in situ formation of DNA-templated Ag2S photoactive materials, accompanied by hybridization chain reaction signal amplification.

Determination of the activity of T4 polynucleotide kinase phosphatase by exploiting the sequence-dependent fluorescence of DNA-templated copper nanoclusters.

A fluorometric method is described for the determination of the activity of the enzyme T4 polynucleotide kinase phosphatase (T4 PNKP). A short 3'-terminus phosphorylated DNA strand is hybridized with a long DNA strand to produce a partially double-stranded DNA (dsDNA) substrate. On addition of T4 PNKP, the substrate is dephosphorylated to generate the long dsDNA, and then the long dsDNA acted as a template for synthesizing copper nanoclusters (CuNCs). The dsDNA-templated CuNCs display fluorescence with excitation/emission peak wavelengths of 340/570 nm. The fluorescence is DNA sequence-dependent. A DNA substrate was designed to enhance fluorescence and to reduce the background in order to improve the sensitivity of the assay. The assay has an analytical range that extends from 0.07 U mL-1 to 15 U mL-1 and a detection limit of 0.06 U mL-1. Graphical abstract The sequence-dependent fluorescence of DNA-templated copper nanoclusters, which are in-situ synthesized through the reduction of CuSO4 by ascorbate (Vc) in the presence of dsDNA template, is utilized to obtain the method for sensitive detection of T4 PNKP activity with near-zero background.

Construction of a thermo-sensitive pRI857 vector for efficient DNA capturing in Escherichia coli.

OBJECTIVES: To establish a positive cloning system with a zero background for high-throughput DNA cloning purpose. RESULTS: The cloning vector, pRI857, and the genomic-library construction vector, pRI857-BAC, were constructed based on the mechanism of expression of the thermo-sensitive cI857 repressor gene that can stringently repress the PR promoter and kanamycin resistance gene (PR-kan R ) at 30 °C, but have no effect on PR-kan R gene at 37 °C or at higher temperatures. When the pRI857 vectors were transformed into E. coli with or without a target foreign DNA fragment inserted at the BfrBI site of the cI857 gene, only colonies with the foreign DNA fragment survive. We extended this method to construct a pRI857-BAC vector for genomic library cloning which displays an efficiency of ~107 cfu per µg of genomic DNA, with no empty vectors detected. CONCLUSIONS: Cloning by indirect activation of resistance marker gene represents a novel DNA-capturing system, which can be widely applied for high-throughput DNA cloning.

Zero-Background Helicase-Dependent Amplification and Its Application to Reliable Assay of Telomerase Activity in Cancer Cell by Eliminating Primer-Dimer Artifacts.

Primer-dimer artifacts resulting from unintended template-independent primer-primer interactions often hinder the specific amplification of nucleic acids. We demonstrate, for the first time, zero-background helicase-dependent amplification (HDA), with low concentrations of both ATP and dNTPs. This strategy achieved the reliable evaluation of telomerase activity in cancer cells by eliminating primer-dimer artifacts, which have plagued many previous methods with reduced specificity. We found that the performance of the telomerase assay by zero-background HDA was negatively affected by highly concentrated cellular proteins. This inhibitory effect is attributed to the binding of DNA templates to proteins, thus making them unavailable for polymerases. However, gold nanoparticles were demonstrated to highly attenuate such inhibition by abundant proteins, and to enhance the assay sensitivity and reliability when the reaction was performed with concentrated cell extracts.

Near zero background noise photoelectrochemical sensor based on sensitized signal probe CdS QDs-Dox intercalating double-stranded DNA for PEDV detection.

The highly sensitive detection method for porcine epidemic diarrhea virus (PEDV) is crucial for promptly identify infected pigs and effectively control the spread of the virus. In this study, the sensitization enhancement of organic photoactive material was combined with near zero background noise strategy for PEDV sensitive detection. A novel sensitized signal probe CdS quantum dots-doxycycline complex (CdS QDs-Dox) was prepared serving as a photoelectrochemical (PEC) probe embedded in dsDNA. Subsequently, a thiol-modified upstream inner primer (SH-FIP) was immobilized on the surface of electrode modified with gold nanoparticles (Au NPs) via Au-S bonding, enabling the loop-mediated isothermal amplification (LAMP) of PEDV on the electrode surface. The PEC probe (CdS QDs-Dox) embedded in the amplified dsDNA groove showed an increasing photocurrent signal with the rise of PEDV concentration, establishing a near-zero background LAMP-PEC sensing platform for PEDV detection. Under optimized conditions, the photocurrent intensity of this platform exhibited a good linear relationship with PEDV concentrations ranging from 0.0005 pg/µL to 10 pg/µL, achieving a detection limit as low as 0.17 fg/µL. This platform demonstrates outstanding specificity and sensitivity, thereby enabling precise quantitative detection of diverse pathogens.

AuNPs/CdS QDs/CeO2 ternary nanocomposite coupled with scrollable three-dimensional DNA walker mediated cycling amplification for sensitive photoelectrochemical miRNA assay.

Herein, a novel ternary nanocomposite (AuNPs/CdS QDs/CeO2) with excellent photoelectrochemical (PEC) performance was synthesized as signal probe to construct a near-zero background biosensor for sensitive miRNA-182-5p detection, by integrating with a scrollable three-dimensional (3D) DNA walker mediated cleavage cycling amplification. Impressively, the formation and rolling of scrollable 3D DNA walker triggered by target could realize dynamic, rapid and specific digestion of hairpin DNA on electrode with the aid of Exonuclease III (Exo III), which thus exposed abundant binding sites for assembling stable DNA labeled AuNPs/CdS QDs/CeO2 nanoprobes. Thanks to the formation of type-II heterojunction (between CeO2 and CdS QDs) and Schottky junction (generated by CeO2 and AuNPs), an ideal photoelectric conversion efficiency accompanied with stunningly improved photocurrent was thus acquired for significantly improving the detection sensitivity. It turned out that the detection limit (LOD) of biosensor was ultralow (31 aM). Significantly, the proposed PEC biosensor would exhibit great potential for the composite as a splendid indicator and provide an avenue for constructing the sensing platform with excellent sensitivity and ultralow background.

Terminal deoxynucleotidyl transferase combined CRISPR-Cas12a amplification strategy for ultrasensitive detection of uracil-DNA glycosylase with zero background.

A simple and highly sensitive biosensing strategy was reported by cascading terminal deoxynucleotidyl transferase (TdT)-catalyzed substrate extension and CRISPR-Cas12a -catalyzed short-stranded DNA probe cleavage. Such a strategy, which is named as TdT-combined CRISPR-Cas12a amplification, gives excellent signal amplification capability due to the synergy of two amplification steps, and thus shows great promise in the design of various biosensors. Based on this strategy, two representative biosensors were developed by simply adjusting the DNA substrate design. High signal amplification efficiency and nearly zero background endowed the biosensors with extraordinary high sensitivity. By utilizing these two biosensors, ultrasensitive detection of uracil-DNA glycosylase (UDG) and T4 polynucleotide kinase (T4 PNK) was achieved with the detection limit as low as 5 × 10-6 U/mL and 1 × 10-4 U/mL, respectively. The proposed UDG-sensing platform was also demonstrated to work well for the UDG activity detection in cancer cells as well as UDG screening and inhibitory capability evaluation, thus showing a great potential in clinical diagnosis and biomedical research.

Nuclear Resonance Vibrational Spectroscopy: A Modern Tool to Pinpoint Site-Specific Cooperative Processes.

Nuclear resonant vibrational spectroscopy (NRVS) is a synchrotron radiation (SR)-based nuclear inelastic scattering spectroscopy that measures the phonons (i.e., vibrational modes) associated with the nuclear transition. It has distinct advantages over traditional vibration spectroscopy and has wide applications in physics, chemistry, bioinorganic chemistry, materials sciences, and geology, as well as many other research areas. In this article, we present a scientific and figurative description of this yet modern tool for the potential users in various research fields in the future. In addition to short discussions on its development history, principles, and other theoretical issues, the focus of this article is on the experimental aspects, such as the instruments, the practical measurement issues, the data process, and a few examples of its applications. The article concludes with introduction to non-57Fe NRVS and an outlook on the impact from the future upgrade of SR rings.

Zero background and triple-signal amplified fluorescence aptasensor for antibiotics detection in foods.

It's important to eliminate matrix interference for accurate detecting antibiotic residues in complex food samples. In this study, we designed a zero-backgrounded fluorescence aptasensor to achieve on-site detection of antibiotic residues, with chloramphenicol (CAP) as representative analyte. Moreover, a three stir-bars assisted target recycling system (TSBTR) was designed to achieve triple signal amplification and increase the sensitivity. The bars included one magnetic stir-bar modified with two kinds of long DNA chains, and two gold stir-bars modified with Y shape-duplex DNA probes respectively. In the presence of CAP, the target could recurrently react with the probes on the bars and replace a large amount of long DNA chains into supernatant. After then, the bars were taken out and SYBR green dye was added to the solution. The dye can specifically intercalate into the duplex structures of DNA chains to emit fluorescence while not emitting a signal in its free state. Under the optimized experimental conditions, a wide linear response range of 5 orders of magnitude from 0.001 ng mL-1 to 10 ng mL-1 was achieved with a detection limit of 0.033 pg mL-1 CAP. The assay was successfully employed to detect CAP in food samples (milk & fish) with consistent results with ELISA's. High selectivity and sensitivity were attributed to the zero background signal and triple signal-amplification strategy. Moreover, the detection time can be shortened to 40 min due to that three signal amplified process can occur simultaneously. The fluorescent aptasensor was also label- and enzyme-free. All these ensure the platform to be rapid, cost-effective, easily-used, and is especially appropriate for detection antibiotics in food safety.

A two dimensional metal-organic framework nanosheets-based fluorescence resonance energy transfer aptasensor with circular strand-replacement DNA polymerization target-triggered amplification strategy for homogenous detection of antibiotics.

In the study, a novel two dimensional metal-organic framework (Cu-TCPP nanosheets) based fluorescence resonance energy transfer (FRET) aptasensing platform was developed for detecting antibiotics. Cu-TCPP nanosheets were employed for quenching the background fluorescence and circular strand-replacement DNA polymerization (CSRP) for signal amplification. To fulfill the purpose, we designed an aptamer hairpin probe (HP) whose stem can be opened while specifically binding to target. Then the opened HP would bind with the primer. Under the action of polymerase, extension reaction was induced to generate double-stranded DNA (dsDNA), and then the target was released for the next cycle. Finally, SYBR Green I (SG) can bind with dsDNA to produce strong fluorescence response for quantification of target. It's worth mentioning that the fluorescence of HP/SG complex and free SG could be completely quenched by Cu-TCPP nanosheets while that of dsDNA/SG complex wouldn't be affected. Thus, the sensor produced negligible background signals. It can produce 7.5-fold improved S/N compared to a graphene oxide (GO)-based FRET aptasensor. Chloramphenicol (CAP) was chosen as the model analyte to demonstrate the feasibility of the sensor system. The detection range is broad from 0.001 to 10 ng mL-1 with a detection limit down to 0.3 pg mL-1. The proposed assay was label free and can be used in homogenous detection which greatly simplifies the complexity of operations. The strategy opens a new way to develop sensitive, in-situ and simple assay for antibiotics in foods.

Ultrasensitive detection of uranyl by graphene oxide-based background reduction and RCDzyme-based enzyme strand recycling signal amplification.

We proposed a novel strategy which combines graphene oxide-based background reduction with RCDzyme-based enzyme strand recycling amplification for ultrahigh sensitive detection of uranyl. The RCDzyme is designed to contain a guanine (G)-rich sequence that replaces the partial sequence in an uranyl-specific DNAzyme. This multifunctional probe can act as the target recognition element, DNAzyme and the primer of signal amplification. The presence of UO2(2+) can induce the cleavage of the substrate strands in RCDzyme. Then, each released enzyme strand can hybridize with another substrate strands to trigger many cycles of the cleavage by binding uranyl, leading to the formation of more G-quadruplexes by split guanine-rich oligonucleotide fragments. The resulting G-quadruplexes could bind to N-methyl-mesoporphyrin IX (NMM), causing an amplified detection signal for the target uranyl. Next, graphene oxide-based background reduction strategy was further employed for adsorbing free ssDNA and NMM, thereby providing a proximalis zero-background signal. The combination of RCDzyme signal amplification and proximalis zero-background signal remarkably improves the sensitivity of this method, achieving a dynamic range of two orders of magnitude and giving a detection limit down to 86 pM, which is much lower than those of related literature reports. These achievements might be helpful in the design of highly sensitive analytical platform for wide applications in environmental and biomedical fields.