Signal Amplification Research Articles

Nanopore single molecule ATP detection based on the dual effects of specific capturing and signal amplification

Adenosine triphosphate (ATP) plays a crucial role in energy metabolism. Some serious diseases such as Alzheimer’s disease are related to abnormal ATP concentrations. Fast, sensitive, and specific ATP detection is important and challenging in disease diagnosis and biomedical research. In present study, conical nanopore was designed and employed to detect ATP at single molecule level. For overcoming the inherent defects such as non-selectivity and low sensitivity of nanopore in sensing small target molecule, two collaborative improvements of specific identification and signal amplification were proposed. We first used split aptamer capture probes (H1 and S1) to anchor ATP molecule, and then used auxiliary probes (H2 and H3) to form a long-nicked duplex ATP-DNA concatemer by hybridization chain reaction (HCR). Referring ATP-DNA concatemer rather than native ATP to nanopore sensor, characteristic current signals with high signal-to-noise ratio were obtained. By such indirect way, specific and sensitive ATP detection was realized with a linear range from 20 pM to 5 nM and a detection limit of 3.9 pM (S/N = 3). Additionally, the sensor exhibited many merits such as simplify, anti-interference, and especially allowing ATP detection in real sample. So, we believe that the nanopore sensor has good application prospects in screening and management of ATP-related diseases.

Biomimetic cascade intelligent paper chip sensor based on bimetallic porphyrin-based covalent organic framework with triple-enzyme mimetic activities

Enzyme-induced biological cascade catalysis can achieve selective and efficient transformations of substrates in vivo. Thus, the biomimetic cascade systems have attracted much attention in biological detection on account of their ingenious strategies for signal conduction and amplification. In this study, a porphyrin-based covalent organic framework (COFFe/CoP-Ph) with bimetal-nitrogen-coordinate centers (M − N4) were constructed via Schiff base reaction. The synergistic effect of the highly exposed M − N4 bimetallic active sites and the unique topology overcame the self-limitation of nanozyme, resulting in a significant enhancement of the triple-enzyme mimetic activities of COFFe/CoP-Ph, namely oxidase (OXD)-, peroxidase (POD)-, and catalase (CAT)-like activities. Simultaneously, a multiple enzyme-driven cascade catalysis strategy for signal amplification was proposed utilizing the OXD- and POD-like mimetic mechanisms of COFFe/CoP-Ph, enabling the colorimetric determination of malathion without the addition exogenous H2O2. The established colorimetric sensor exhibited an exceptional linear relationship within the range of 1–18 μM and a low detection limit of 11 nM. Subsequently, a portable smartphone platform was developed by integrating paper chip, facilitating successful malathion point-of-care testing (POCT) in real samples. This work not only creates a bimetallic COF nanozyme and establishes an efficient cascade amplification strategy, but also introduces a novel design approach for development of a low-cost and user-friendly POCT method.

Upgraded and Light-Up Biosensing Platform: Entropy-Driven Catalysis Circuit Manipulates the Configuration Transformation of Novel DNA Silver Nanoclusters on the Graphene Oxide Surface.

To tackle the predicament of the traditional turn-off mechanism, exploring an activated turn-on system remains an intriguing and crucial objective in biosensing fields. Herein, a dark DNA Ag nanocluster (NC) with hairpin-structured DNA containing a six-base cytosine loop (6C loop) as a template is atypically synthesized. Intriguingly, the dark DNA Ag NCs can be lit to display strong red-emission nanoclusters. Building upon these exciting findings, an unprecedented and upgraded turn-on biosensing system [entropy-driven catalysis circuit (EDCC)-Ag NCs/graphene oxide (GO)] has been created, which employs an EDCC to precisely manipulate the conformational transition of DNA Ag NCs on the GO surface from adsorption to desorption. Benefiting from the effective quenching of GO and signal amplification capability of the EDCC, the newly developed EDCC-Ag NCs/GO biosensing system displays a high signal-to-background (S/B) ratio (26-fold) and sensitivity (limit of detection as low as 0.4 pM). Meanwhile, it has good specificity, excellent stability, and reliability in both buffer and biological samples. To the best of our knowledge, it is the first example that adopts an EDCC to precisely modulate the configuration transformation of DNA Ag NCs on the GO surface to obtain a biosensor with low background, strong fluorescence, high contrast, and sensitivity. This exciting finding may provide a new route to fabricate a novel turn-on biosensor based on hairpin-templated DNA Ag NCs in the optical imaging and bioanalytical fields.

DNA Polymerase-Endonuclease Efficiently Synthesizes DNA to Prepare DNA Materials and Develop Novel Signal Amplification System.

DNA biosynthesis, a focus of fundamental and applied research, typically involves DNA polymerases by using templates, primers, and dNTPs. Some polymerases can polymerize dNTPs for DNA de novo synthesis, although this is generally to occur randomly. This novel synthesis method has garnered our attention and practical use. Herein, we observed that the addition of endonuclease significantly enhances the efficiency of the de novo synthesis reaction catalyzed by the DNA polymerase. We further investigated the reaction conditions that influence this efficiency. Building on the optimal reaction conditions, we developed a rapid and efficient strategy for preparing DNA hydrogel. Further, coupled with the CRISPR-Cas system, we developed a nucleic acid signal amplification system characterized by versatility, sensitivity, specificity, and no risk of aerosol contamination. We successfully detected viral nucleic acids in clinical samples. In summary, our study demonstrates the significant potential of DNA polymerase- and endonuclease-catalyzed DNA de novo synthesis in diverse applications.

Site-Directed Electrochemical Grafting for Amplified Detection of Antibody Pharmaceuticals.

Antibody pharmaceuticals have become the most popular immunotherapeutic drugs and are often administered with low serum drug dosages. Hence, the development of a highly sensitive method for the quantitative assay of antibody levels is of great importance to individualized therapy. On the basis of the dual signal amplification by the glycan-initiated site-directed electrochemical grafting of polymer chains (glyGPC), we report herein a novel strategy for the amplified electrochemical detection of antibody pharmaceuticals. The target of interest was affinity captured by a DNA aptamer ligand, and then the glycans of antibody pharmaceuticals were decorated with the alkyl halide initiators (AHIs) via boronate cross-linking, followed by the electrochemical grafting of the ferrocenyl polymer chains from the glycans of antibody pharmaceuticals through the electrochemically controlled atom transfer radical polymerization (eATRP). As the glycans can be decorated with multiple AHIs and the grafted polymer chains are composed of tens to hundreds of electroactive tags, the glyGPC-based strategy permits the dually amplified electrochemical detection of antibody pharmaceuticals. In the presence of trastuzumab (Herceptin) as the target, the glyGPC-based strategy achieved a detection limit of 71.5 pg/mL. Moreover, the developed method is highly selective, and the results of the quantitative assay of trastuzumab levels in human serum are satisfactory. Owing to its uncomplicated operation and cost-effectiveness, the glyGPC-based strategy shows great promise in the amplified electrochemical detection of antibody pharmaceuticals.

Au nanodisks-based 2D photonic crystals fabricated by single-beam laser interference lithography: A simple and reliable alternative for highly efficient large-scale SERS molecular sensors

Noble metal-based Photonic Crystals (PCs) have emerged as outstanding candidates for precise light management, projecting applications in strategic areas for society like high-sensitivity and fast molecular (inorganic/organic/bio) sensing by Surface-Enhanced Raman Spectroscopy (SERS). In this work, we report an exhaustive study on the potential of large-scale (active area >1 [cm2]) Au nanodisks-based 2D PCs fabricated by single-beam Laser Interference Lithography (LIL) for high-performance SERS molecular sensing. This technique was used to fabricate periodic nanoarrays (period of 470 [nm]) of Au nanodisks with thicknesses from 50 up to 125 [nm]. The period was chosen following Finite-Difference Time-Domain (FDTD) simulations that suggested the best electric-near field enhancement for this condition. Confocal Raman microscopy and Methylene Blue (MB) as active Raman marker, were used to assess the samples' performance for molecular sensing. SERS studies have shown that the nanodisks' thickness can be a considerable size parameter for the Raman signal amplification, observing higher signal enhancements for higher thicknesses. The observed thickness effects on the Raman signal enhancement were consistent with FDTD simulations, which predicted higher electric-near field amplifications for higher thickness within the red/near-infrared range. Results show that our PCs enable to measure the characteristic Raman footprint of the analyte with good spectral resolution using relatively low powers (0.04–1 [mW]) and short acquisition times (1–30 [s]), considering an MB surface mass density as low as 2.6 [ng/cm2]. SERS enhancement factors as high as 2 x 107 were achieved for PCs with the highest thickness, representing a competitive performance concerning typically reported values (104–107) for current noble metal-based PCs technologies and a new record concerning PCs fabricated by LIL (104–105). This research demonstrates the high competitivity of these simple Au nanodisks-based 2D PCs, fabricated using an efficient large-scale and low-cost lithography technique, for fast, high spectral resolution and highly reproducible SERS-based molecular sensing.

Exo I-based cyclic digestion coupled with synergistic enhancement strategy for integrating dual-mode optical aptasensor platform

The improvement of dual-mode techniques was of particular interest to researchers, which might enhance the detection performance and applicability. Here, a dual-mode optical aptasensor (DO-aptasensor) platform based on exonuclease I (Exo I) cyclic digestion and synergistic enhancement strategy had proposed for zearalenone (ZEN). Following the preparation of dumbbell-shaped signal probe, the Exo I-based cyclic digestion amplification performed, and then the synergistic enhancement effect carried out to achieve the Poly-HRP-based colorimetry and FAM-SGI-based fluorescence. The efficient homogeneous system realized through the magnetic separation, while the signal interference further eliminated by the graphene oxide (GO). The LOD values were as low as 0.067 ng mL−1 for colorimetry mode and 0.009 ng mL−1 for fluorescence mode, which reduced 23-fold and 172-fold than ELASA by same ZEN-Apt. This promising platform gave rise to a dual-mode optical readout, improved sensitivity and positively correlated detection. Meanwhile, the DO-aptasensor also exhibited the acceptable specificity, desirable reliability and excellent practicability. This novel avenue of aptasensor platform hold great potential for dual-mode optical monitoring of other targets, which can further expand the application scope of Exo I-based signal amplification and synergistic enhancement effect.

Photoactivated Controlled Dnazyme Platform for on-Demand Activation Sensitive Electrochemiluminescence mRNA Analysis.

Programming ultrasensitive and stimuli-responsive DNAzyme-based probes holds great potential for on-demand biomarker detection. Here, an optically triggered DNAzyme platform was reported for on-demand activation-sensitive electrochemiluminescence (ECL) c-myc mRNA analysis. In this design, the sensing and recognition function of the split DNAzyme (SDz) probe was silent by engineering a blocking sequence containing a photocleavable linker (PC-linker) group at a defined site that could be indirectly cleaved by 302 nm ultraviolet (UV) light. When the SDz probes were assembled on the Au nanoparticles and potassium (K) element doped graphitic carbon nitride nanosheet (K-doped g-C3N4) covered electrode, UV light activation induces the configurational switching and consequently the formation of an active DNAzyme probe with the help of target c-myc mRNA, allowing the cleavage of the substrate strand by magnesium ions (Mg2+). Thus, the release of a ferrocene (Fc)-labeled DNAzyme 2 strand contributed to an extreme ECL signal recovery. In the meantime, the released target c-myc mRNA combined another inactive SDz motif to form active DNAzyme and repeat the cyclic cleavage reaction, resulting in the signal amplification. Furthermore, according to the responses toward two other designed nPC-SDz and m-SDz probes, we demonstrated that controlled UV light mediated photoactivation of the DNAzyme biosensor "on demand" effectively constrained the ECL signal to the mRNA of interest. Moreover, false positive signals could also be avoided due to such a photoactivation design with UV light. Therefore, this study provided a simple methodology that may be broadly applicable for investigating the mRNA-associated physiological events that were difficult to access using traditional DNAzyme probes.

Double base mismatches mediated catalytic hairpin assembly for enzyme-free single-base mutation detection: integrating signal recognition and amplification in one.

Single nucleotide polymorphism (SNP) biosensors are emerging rapidly for their promising applications in human disease prevention diagnosis, treatment, and prognosis. However, it remains a bottleneck in equipping simple and stable biosensors with the traits of high sensitivity, non-enzyme, and low cost. Double base mismatches mediated chain displacement reactions have attracted fascinating advantages of tailorable thermodynamics stability, non-enzyme, and excellent assembly compliance to involvement in SNP identification. As the base mismatch position and amount in DNA sequence can be artificially adjusted, it provides plenty of selectivity and specificity for exploring perfect biosensors. Herein, abiosensor with double base mismatches mediated catalytic hairpin assembly (CHA) is designed via one base mismatch in the toehold domain and the other base mismatch in the stem sequence of hairpin 1 (H1) by triggering CHA reaction to achieve selective amplification of the mutation target (MT) and fluorescence resonance energy transfer (FRET) effect that is composed of Cy3 and Cy5 terminally attached H1 and hairpin 2 (H2). Depending on the rationally designed base mismatch position and toehold length, the fabricated biosensors show superior SNP detection performance, exhibiting a good linearity with high sensitivity of 6.6 fM detection limitand a broad detection abundance of 1%. The proposed biosensor can be used to detect the KRAS mutation gene in real samples and obtain good recoveries between 106 and 116.99%. Remarkably, these extendible designs of base mismatches can be used for more types of SNP detection, providing flexible adjustment based on base mismatch position and toehold length variations, especially for their thermodynamic model for DNA-strand displacement reactions.

A smartphone-based fluorescent biosensor with metal-organic framework biocomposites and cotton swabs for the rapid determination of tetrodotoxin in seafood

BackgroundTetrodotoxin (TTX) is a potent neurovirulent marine biotoxin that is present in puffer fish and certain marine animals. It is capable of causing severe neurotoxic symptoms and even death when consumed through contaminated seafood. Due to its high toxicity, developing an effective assay for TTX determination in seafood has significant benefits for food safety and human health. Currently, it remains challenging to achieve on-site determination of TTX in seafood. To facilitate mass on-site assays, more affordable technologies utilizing accessible equipment that require no skilled personnel are needed. ResultsA smartphone-based portable fluorescent biosensor is proposed for TTX determination by using metal-organic framework (MOF) biocomposites and cotton swabs. Oriented antibody (Ab)-decorated and fluorescent quantum dot (QD)-loaded MOF biocomposites (QD@MOF*Ab) are rapidly synthesized for binding targets and fluorescent responses by utilizing the tunability of zinc-based MOF. Moreover, facile Ab-immobilized household cotton swabs are utilized as TTX capture tools. TTX forms sandwich immune complexes with QD@MOF*Ab probes, achieving signal amplification. These probes are excited by a portable device to generate bright fluorescent signals, which can be detected by the naked eye, and TTX quantitative results are obtained using a smartphone. When observed with the naked eye, the limit of detection (LOD) is 0.4 ng/mL, while intelligent quantitation presents an LOD of 0.13 ng/mL at logarithmic concentrations of 0.2–400 ng/mL. SignificanceThis biosensor is convenient to use, and an easy-to-operate analysis is completed within 15 min, thus demonstrating excellent performance in terms of detection speed and portability. Furthermore, it successfully determines TTX contents in puffer fish and clam samples, demonstrating its potential for monitoring seafood. Herein, this work provides a favorable rapid sensing platform that is easily portable.

Thymine-Hg2+-Thymine strategy in MOF-based electrochemical aptamer sensor for PAEs detection

Phthalate acid esters (PAEs), known food contaminants, can lead to reproductive problems, respiratory diseases, childhood obesity, and neuropsychological disorders. This work aims to develop an electrochemical aptamer sensor for the detection of DAP and its analogs. To achieve specific recognition ability for PAEs, we have introduced the thymidine-Hg2+-thymidine strategy for the first time in an electrochemical aptamer sensor, incorporating metal–organic frameworks (MOFs) as the carrier for the aptamer. Additionally, nano signal amplification technology was applied by immobilizing MXene on the electrode to intensify the sensor’s current signal. In the presence of PAEs, these molecules specifically bound to the aptamers, resulting in competitive release of Hg2+ and aptamers connected to Hg2+, subsequently reducing current signaling. Under optimal experimental conditions, the designed sensor achieved a linear range from 1.65 × 10−5 mg/mL to 3 × 10−3 mg/mL and an extremely low detection limit of 8.94 × 10−6 mg/mL. Furthermore, it exhibited good reliability and practicality in detecting actual samples.

Detection of fucosylated extracellular vesicles miR-4732-5p related to diagnosis of early lung adenocarcinoma by the electrochemical biosensor

Our preliminary investigation has identified the potential of serum fucosylated extracellular vesicles (EVs) miR-4732-5p in the early diagnosis of lung adenocarcinoma (LUAD) by a fucose-captured strategy utilizing lentil lectin (LCA)-magnetic beads and subsequent screening of high throughput sequencing and validation of real-time quantitative polymerase chain reaction (RT-qPCR). Considering the relatively complicated procedure, expensive equipment, and stringent laboratory condition, we have constructed an electrochemical biosensor assay for the detection of miR-4732-5p. miR-4732-5p is extremely low in serum, down to the fM level, so it needs to be detected by highly sensitive electrochemical methods based on the Mg2+-dependent DNAzyme splitting nucleic acid lock (NAL) cycle and hybridization chain reaction (HCR) signal amplification. In this study, signal amplification is achieved through the dual amplification reactions using NAL cycle in combination with HCR. In addition, hybridized DNA strands bind to a large number of methylene blue (MB) molecules to enhance signaling. Based on the above strategy, we further enhance our signal amplification strategies to improve detection sensitivity and accuracy. The implementation of this assay proceeded as follows: initially, miR-4732-5p was combined with NAL, and then Mg2+-dependent DNAzyme splitted NAL to release auxiliary DNA (S1) strands, which were subsequently captured by the immobilized capture probe DNA (C1) strands on the electrode surface. Following this, abundant quantities of DNA1 (H1) and DNA2 (H2) tandems were generated by HCR, and S1 strands then hybridized with the H1 and H2 tandems through base complementary pairing. Finally, MB was bonded to the H1 and H2 tandems through π–π stacking interaction, leading to the generation of a signal current upon the detection of a potential capable of inducing a redox change of MB by the electrode. Furthermore, we evaluated the performance of our developed electrochemical biosensor assay. The results demonstrated that our assay is a reliable approach, characterized by its high sensitivity (with a detection limit of 2.6 × 10−17 M), excellent specificity, good accuracy, reproducibility, and stability. Additionally, it is cost-effective, requires simple operation, and is portable, making it suitable for the detection of serum fucosylated extracellular vesicles miR-4732-5p. Ultimately, this development has the potential to enhance the diagnostic efficiency for patients with early-stage LUAD.

Peripheral preprocessing in Drosophila facilitates odor classification

The mammalian brain implements sophisticated sensory processing algorithms along multilayered ("deep") neural networks. Strategies that insects use to meet similar computational demands, while relying on smaller nervous systems with shallow architectures, remain elusive. Using Drosophila as a model, we uncover the algorithmic role of odor preprocessing by a shallow network of compartmentalized olfactory receptor neurons. Each compartment operates as a ratiometric unit for specific odor-mixtures. This computation arises from a simple mechanism: electrical coupling between two differently sized neurons. We demonstrate that downstream synaptic connectivity is shaped to optimally leverage amplification of a hedonic value signal in the periphery. Furthermore, peripheral preprocessing is shown to markedly improve novel odor classification in a higher brain center. Together, our work highlights a far-reaching functional role of the sensory periphery for downstream processing. By elucidating the implementation of powerful computations by a shallow network, we provide insights into general principles of efficient sensory processing algorithms.

Total losses and dispersion effects management and upgrading fiber reach in ultra-high optical transmission system based on hybrid amplification system

Abstract This work demonstrated the total losses and dispersion effects management and upgrading the fiber reach in ultra-high optical transmission system based on hybrid amplification system. Based on the requirements of the system, a number of trade-offs must be made between the numerous interconnected performance characteristics of each component in the design of a high quality transmission link. The transmission distance is the greatest distance that an optical signal has to travel. It affects the choice of optical component, fiber type, and signal amplification techniques. Fiber amplifiers or signal regeneration may be required to compensate for signal loss over greater distances. Reviewing these crucial aspects helps when selecting the best optical components and system layout. This ensures the optical link performs as expected. Hybrid amplification system unit (EDFA/Raman/EDFA), (EDFA/Raman), (BFA/PDFA) and (Raman/EDFA) is employed in the transmission system to strength the signal and upgrade the system performance operation efficiency. Space division multiplexing scheme is also employed with the propagation scheme in order to upgrade and control of the total losses and dispersion effects. The optimum total losses/dispersion/repeater spacing are studied with the variations of the total number of links per fiber core channel.

Preamplification-free ultra-fast and ultra-sensitive point-of-care testing via LwaCas13a

CRISPR based nucleic acid detection technology provides a deployable approach to point of care testing. While, there remain challenges limiting its practical applications, such as the need for pre-amplification and the long turnaround time. Here, we present a self-cascade signal amplification method with LwaCas13a and an artificially designed “U” rich RNA of stem-loop structure (URH) for pre-amplification-free ultra-fast and ultra-sensitive point-of-care testing (PASSPORT). The PASSPORT system contains: URH, crRNA targeted the URH, crRNA targeted the interesting RNA, fluorescent RNA reporter and LwaCas13a. The assay realized the detection of 100 copies/mL, within 5 min. The PASSPORT platform was further adopted for the detection of marker gene from SASR-CoV-2 and Severe fever with thrombocytopenia syndrome virus (SFTSV), respectively, and 100% accuracy for the analysis of clinical specimens (100 SASR-CoV-2 specimens and 16 SFTSV specimens) was obtained. Integrated with a lateral flow assay device, this assay could provide an alternative platform for the development of point of care testing (POCT) biosensors. PASSPORT has the potential to enable sensitive, specific, user-friendly, rapid, affordable, equipment-free and point-of-care testing for the purpose of large-scale screening and in case of epidemic outbreak.

Target recognition-initiated allosteric probe-based multiple signal amplification strategy for sensitive and direct Pseudomonas aeruginosa detection

Target recognition-initiated allosteric probe-based multiple signal amplification strategy for sensitive and direct Pseudomonas aeruginosa detection

Enhanced spontaneous radiation of quantum dots based on modulated anapole states in dielectric metamaterial

Dielectric nanostructures exhibit low-loss electrical and magnetic resonance, making them ideal for quantum information processing. In this study, the periodic double-groove silicon nanodisk (DGSND) is used to support the anapole state. Based on the distribution properties of the electromagnetic field in anapole states, the anapoles are manipulated by cutting the dielectric metamaterial. Quantum dots (QDs) are used to stimulate the anapole and control the amplification of the photoluminescence signal within the QDs. By opening symmetrical holes in the long axis of the nanodisk in the dielectric metamaterial, the current distribution of Mie resonance can be adjusted. As a result, the toroidal dipole moment is altered, leading to an enhanced electric field (E-field) and Purcell factor. When the dielectric metamaterial is deposited on the Ag substrate separated by the silicon dioxide (SiO2) layer, the structure exhibits ultra-narrow perfect absorption with even higher E-field and Purcell factor enhancement compared to silicon (Si) nanodisks.

Oxide semiconductor based deep‐subthreshold operated read‐out electronics for all‐printed smart sensor patches

AbstractThe ability to fabricate an entire smart sensor patch with read‐out electronics using commercial printing techniques may have a wide range of potential applications. Although solution‐processed oxide thin film transistors (TFTs) are capable of providing high mobility electron transport, resulting in large ON‐state current and power output, there is hardly any literature report that uses the printed oxide TFTs at the sensor interfaces. Here, printed amorphous indium‐gallium‐zinc oxide (a‐IGZO)‐based deep‐subthreshold operated TFTs that comprise signal amplifiers and analog‐to‐digital converters (ADCs) that can successfully digitalize the analog sensor signals up to a frequency range of 1 kHz are reported. In addition, exploiting the high current oxide TFTs, a current drive circuit placed after the ADC unit has been found useful in producing easy‐to‐detect visual recognition of the sensor signal at a predefined threshold crossover. Notably, the entire smart sensor patch is demonstrated to operate at a low supply voltage of ≤2 V, thereby ensuring that it can be an on‐chip energy source compatible and standalone detection unit.

DNA Walker-Driven Mass Nanotag Assembly System for Simultaneously Profiling Dual Markers of Oxidative Stress at Different Cellular Locations.

Simultaneous profiling of redox-regulated markers at different cellular sublocations is of great significance for unraveling the upstream and downstream molecular mechanisms of oxidative stress in living cells. Herein, by synchronizing dual target-triggered DNA machineries in one nanoentity, we engineered a DNA walker-driven mass nanotag (MNT) assembly system (w-MNT-AS) that can be sequentially activated by oxidative stress-associated mucin 1 (MUC1) and apurinic/apyrimidinic endonuclease 1 (APE1) from plasma membrane to cytoplasm and induce recycled assembly of MNTs for multiplex detection of the two markers by matrix-assisted laser desorption ionization mass spectrometry (MALDI MS). In the working cascade, the sensing process governs the separate activation of w-MNT-AS by MUC1 and APE1 in diverse locations, while the assembly process contributes to the parallel amplification of the ion signal of the characteristic mass tags. In this manner, the differences between MCF-7, HeLa, HepG2, and L02 cells in membrane MUC1 expression and cytoplasmic APE1 activation were fully characterized. Furthermore, the oxidative stress level and dynamics caused by exogenous H2O2, doxorubicin, and simvastatin were comprehensively demonstrated by tracking the fate of the two markers across different cellular locations. The proposed w-MNT-AS coupled MS method provides an effective route to probe multiple functional molecules that lie at different locations while participating in the same cellular event, facilitating the mechanistic studies on cellular response to oxidative stress and other disease-related cellular processes.

15N Hyperpolarization of Metronidazole Antibiotic in Aqueous Media Using Phase-Separated Signal Amplification by Reversible Exchange with Parahydrogen.

Metronidazole is a prospective hyperpolarized MRI contrast agent with potential hypoxia sensing utility for applications in cancer, stroke, neurodegenerative diseases, etc. We demonstrate a pilot procedure for production of ∼30 mM hyperpolarized [15N3]metronidazole in aqueous media by using a phase-separated SABRE-SHEATH hyperpolarization method, with nitrogen-15 polarization exceeding 2.2% on all three 15N sites achieved in less than 2 min. The 15N polarization T1 of ∼12 min is reported for the 15NO2 group at the clinically relevant field of 1.4 T in the aqueous phase, demonstrating a remarkably long lifetime of the hyperpolarized state. The produced aqueous solution of [15N3]metronidazole that contained only ∼100 μM of residual Ir was deemed biocompatible via validation through the MTT colorimetric test for assessing cell metabolic activity using human embryotic kidney HEK293T cells. This low-cost and ultrafast hyperpolarization procedure represents a major advance for the production of a biocompatible HP [15N3]metronidazole (and potentially other hyperpolarized drugs) formulation for MRI sensing applications.