High Signal Amplification Research Articles

A Comprehensive Review: Versatile Imaging Probe Based on Chemical Materials for Biomedical Applications.

Imaging probe and contrast agents play significant role in combating cancer. Based on special chemical materials, imaging probe can convert cancer symptoms into information-rich images with high sensitivity and signal amplification, accompanying with detection, diagnosis, drug delivery and treatment. In the paper, some inorganic and organic chemical materials as imaging probe, including Ultrasound imaging (US), Optical imaging (OP), Photoacoustic imaging (PA), X-ray Computed Tomography (CT), Magnetic Resonance imaging (MRI), Radionuclide imaging (RNI) probe, as well as multi-modality imaging probe for diagnosis and therapy of tumour were introduced. The sophisticated and comprehensive chemical materials as imaging probe were highlighted in detail. Meanwhile, the advantages and disadvantages of the imaging probe were compared. In order to provide some reference and help researchers for construction imaging probe for tumour diagnosis and treatment, it attempts to exhaustively cover the whole field. Finally, the prospect and challenge for imaging probe were discussed.

Ultrasensitive detection of mycotoxins using a novel single-Atom, CRISPR/Cas12a-Based nanozymatic colorimetric biosensor

Aflatoxin B1 (AFB1) is a typical mycotoxin in cereals, posing a significant threat to the ecology and public health. Overall, the development of nanozymatic sensors with good specificity, high sensitivity, and a clear catalytic mechanism seems to be a challenge for CRISPR/Cas detection of non-nucleic acid targets. In this study, a series of Fe-N-C single-atom enzymes (SAzymes) and Fe-Co magnetic nanoparticles (MNPs) were synthesized via pyrolysis. Fe-N-C SAzymes with high peroxidase-mimetic activity were successfully utilized as TMB chromogenic catalysts for detecting AFB1 via the CRISPR/Cas12a colorimetric aptamer sensor. In the absence of the AFB1 target, its aptamer bound to crRNA and activated the trans-cleavage activity of Cas12a, indiscriminately cutting single-stranded DNA and releasing significant amounts of Fe-N-C SAzymes into the supernatant, which catalyzed the change of TMB to blue. The synthesized Fe-Co MNPs not only effectively reduced the background signal but also significantly reduced the assay cost. This study combined SAzymes with CRISPR/Cas12a technology for the first time for non-nucleic acid target detection. The combination of Fe-N-C SAzymes’ high catalytic activity and stability, and the CRISPR/Cas12a system’s high specificity and signal amplification, significantly improved the detection system’s sensitivity and specificity, enabling the detection of other targets by substituting the aptamers. Additionally, the potential active sites and catalytic mechanism of Fe-N-C SAzymes were investigated using extended X-ray absorption fine structure (EXAFS) spectroscopy and density functional theory (DFT) simulations. This scheme provides a general method for constructing high-density Fe-N-C SAzymes and offers both experimental and theoretical guidance for understanding their catalytic mechanism.

Cooperative amplification of Prussian blue as a signal indicator and functionalized metal-organic framework-based electrochemical biosensor for an ultrasensitive HE4 assay

Human epididymis protein 4 (HE4), a diagnostic biomarker of ovarian cancer, is crucial for monitoring the early stage of the disease. Hence, it is highly important to develop simple, inexpensive, and user-friendly biosensors for sensitive and quantitative HE4 assays. Herein, a new sandwich-type electrochemical immunosensor based on Prussian blue (PB) as a signal indicator and functionalized metal-organic framework nanocompositesas efficient signal amplifiers was fabricated for quantitative analysis of HE4. In principle, ketjen black (KB) and AuNPs modified on TiMOF (TiMOF-KB@AuNPs) could accelerate electron transfer on the electrode surface and act as a matrix for the immobilization of antibodies via cross-linking to improve the determination sensitivity. The PB that covalently binds to labeled antibodies endows the biosensors with intense electrochemical signals. Furthermore, the concentration of HE4 could be indirectly detected by monitoring the electroactivity of PB. Benefiting from the high signal amplification ability of the PB and MOF nanocomposites, this strategy displayed a wide linear range (0.1–80 ng mL−1) and a lower detection limit (0.02 ng mL−1). Hence, this study demonstrated great promise for application in clinical ovarian cancer diagnosis and treatment, and provided a new platform for detecting other cancer biomarkers.

Activity-Based Fluorescence Diagnostics for Cancer.

Fluorescence imaging is one of the most promising approaches to achieve intraoperative assessment of the tumor/normal tissue margins during cancer surgery. This is critical to improve the patients' prognosis, and therefore various molecular fluorescence imaging probes have been developed for the identification of cancer lesions during surgery. Among them, "activatable" fluorescence probes that react with cancer-specific biomarker enzymes to generate fluorescence signals have great potential for high-contrast cancer imaging due to their low background fluorescence and high signal amplification by enzymatic turnover. Over the past two decades, activatable fluorescence probes employing various fluorescence control mechanisms have been developed worldwide for this purpose. Furthermore, new biomarker enzymatic activities for specific types of cancers have been identified, enabling visualization of various types of cancers with high sensitivity and specificity. This Review focuses on recent advances in the design, function and characteristics of activatable fluorescence probes that target cancer-specific enzymatic activities for cancer imaging and also discusses future prospects in the field of activity-based diagnostics for cancer.

Twice-walk strategy based on three-dimensional DNA walking machine driven by duplex-specific nuclease.

A twice-walk strategy based on a three-dimensional (3D) cleat-equipped DNA walking machine with a high signal amplification efficiency was investigated for ultrasensitive detection of miRNA. Impressively, addition of duplex-specific nuclease (DSN) just once drove the twice-walk strategy, making the strategy simpler. With the advantages of being simple, rapid and ultrasensitive, the biosensor offers potential for use in early clinical diagnosis.

Inkjet‐Printed, Wafer‐Scale Organic Schottky‐Gate Transistors toward Single‐Battery‐Driven Integrated Logic

AbstractOrganic field‐effect transistors switched by insulated gates are the most essential building blocks, while usually plagued with degraded gate control arising from complicated dielectric engineering. Subsequently, the resulting large supply voltage and power consumption remain an essential issue for portable electronics driven by a single battery of only 1.5 V. Herein, wafer‐scale organic Schottky‐gate transistor arrays using inkjet‐printed few‐layer organic semiconducting crystals are reported. The transistors exhibit steep switching characteristics with an average subthreshold swing of 55 mV per dec and high signal amplification efficiency over 45 S A‐1, attributed to efficient Schottky gating and enhanced charge injection. Thereafter, high‐gain inverters are successfully demonstrated with an ultralow power consumption of only 800 pW; also, they are integrated as 1 V driven sequential logic circuits. A coplanar double‐gate geometry is also introduced for low‐voltage, single‐device AND logic. Therefore, the work opens new avenues toward the sustainable advancement in single‐battery‐driven, ultralow‐power organic electronics.

Paper-based lipid bilayer magnetic interface for ultrasensitive assay of tumor-derived extracellular vesicles PD-L1

Programmed death ligand 1 (PD-L1) of tumor-derived extracellular vesicles (T-EVs) is an important biomarker to predict immunotherapy response. The traditional bioassays of T-EVs PD-L1 are limited by the detection specificity because of the distribution of the signals by PD-L1 from nontumor-derived EVs, which leads to unsatisfactory predictive accuracy. In this work, we developed a simple and ultrasensitive method for the quantification of T-EVs PD-L1 by using the lipid bilayer magnetic interface as the isolation and enrichment carrier along with hybridization chain reaction (HCR) for signal amplification in a paper-based device. First, magnetic beads were covered by liposome bilayers and then anchored with the cholesterol modified epithelial cell adhesion molecule (EpCAM) aptamer to construct a fluid multivalent magnetic interface and realize the high-performance isolation of T-EVs. Moreover, the biosensor relied on the PD-L1 aptamer to initiate the HCR of EVs. Benefiting from the high isolation ability of the lipid bilayer magnetic interface and the high signal amplification ability of HCR, the dual-aptamer-based biosensor is capable of highly sensitive detection of T-EVs with a detection limit as low as 5.0 EVs·µL−1 in a paper-based device. Overall, the paper-based lipid bilayer magnetic interface biosensor not only can serve as an alternative tool for predicting the immunotherapy response but also displays great potential in point-of-care disease diagnosis.

Efficient Intracellular MicroRNA Imaging Based on the Localized and Self-Sustainable Catalytic DNA Assembly Circuit.

Building dynamic biological networks, especially DNA circuits, has provided a powerful prospect for exploring the intrinsic regulation processes of live cells. Nevertheless, for efficient intracellular microRNA analysis, the available multi-component circuits are constrained by their limited operating speed and efficiency due to the free diffusion of reactants. Herein, we developed an accelerated Y-shaped DNA catalytic (YDC) circuit for high-efficiency intracellular imaging of microRNA. By grafting the catalytic hairpin assembly (CHA) reactants into an integrated Y-shaped scaffold, the CHA probes were concentrated in a compact space, thus achieving high signal amplification. Profiting from the spatially confined reaction and the self-sustainably assembled DNA products, the YDC system facilitated reliable and in situ microRNA imaging in live cells. Compared with the homogeneously dispersed CHA reactants, the integrated YDC system could efficiently promote the reaction kinetics as well as the uniform delivery of CHA probes, thus providing a robust and reliable analytical tool for disease diagnosis and monitoring.

Ultrasensitive photoelectrochemical biosensor amplified by target induced assembly of cruciform DNA nanostructure for the detection of dibutyl phthalate

In this work, an ultra-sensitive signal quenched photoelectrochemical (PEC) aptasensor for dibutyl phthalate (DBP) detection was constructed by using a target induced cruciform DNA structure as signal amplifier and g-C3N4/SnO2 composite as signal indicator. Impressively, the designed cruciform DNA structure shows high signal amplification efficiency due to the reduced reaction steric hindrance because of its mutually separated and repelled tails, multiple recognition domains, and a fixed direction for the sequential identification of the target. Therefore, the fabricated PEC biosensor demonstrated a low detection limit of 0.3 fM for DBP in a wide linear range of 1 fM to 1 nM. This work offered a novel nucleic acid signal amplification approach for enhancing the sensitivity of PEC sensing platforms for the detection of phthalates (PAEs)-based plasticizer, laying the foundation for its utilization in the determine of real environmental pollutants.

Dual signal amplified electrochemical aptasensor based on PEI-functionalized GO and ROP for highly sensitive detection of cTnI

Cardiac troponin I (cTnI) is considered as the gold standard for the diagnosis of acute myocardial infarction (AMI) because of its excellent specificity and sensitivity. Herein, a novel aptasensor based on the dual signal amplification strategy of Polyethyleneimine functionalized Graphene oxide (GO) and ring-opening polymerization (ROP) for the first time was successfully constructed to achieve high sensitivity detection of cTnI. Briefly, cTnI-aptamer 1 (Apt1) was immobilized on the surface of gold electrode by self-assembly of Au-S bonds to specifically capture cTnI. After specific recognition of cTnI, Apt2 coated PEI-functionalized GO composites acted as macroinitiators for the subsequent ROP reaction. Next, α-amino acid-N-carboxylic acid anhydride ferrocene derivatives (NCA-Fc), the monomer for ROP reaction, was added to the electrode surface. The combined application of PEI-functionalized GO and NCA-Fc better achieves the high sensitivity and signal amplification of the aptasensor. Under optimal conditions, the aptasensor exhibited a wide linear range of 10 fg mL−1 to 10 ng mL−1 and the limit of detection was 3.78 fg mL−1. Moreover, this method displayed the advantages of good selectivity, simple operation and excellent stability. Meanwhile, the aptasensor had good accuracy and applicability even in real serum samples analysis, demonstrating its considerable application potential in biomedical assays.

Self-Stacking Autocatalytic Molecular Circuit with Minimal Catalytic DNA Assembly.

Isothermal autocatalytic DNA circuits have been proven to be versatile and powerful biocomputing platforms by virtue of their self-sustainable and self-accelerating reaction profiles, yet they are currently constrained by their complicated designs, severe signal leakages, and unclear reaction mechanisms. Herein, we developed a simpler-yet-efficient autocatalytic assembly circuit (AAC) for highly robust bioimaging in live cells and mice. The scalable and sustainable AAC system was composed of a mere catalytic DNA assembly reaction with minimal strand complexity and, upon specific stimulation, could reproduce numerous new triggers to expedite the whole reaction. Through in-depth theoretical simulations and systematic experimental demonstrations, the catalytic efficiency of these reproduced triggers was found to play a vital role in the autocatalytic profile and thus could be facilely improved to achieve more efficient and characteristic autocatalytic signal amplification. Due to its exponentially high signal amplification and minimal reaction components, our self-stacking AAC facilitated the efficient detection of trace biomolecules with low signal leakage, thus providing great clinical diagnosis and therapeutic assessment potential.

Sensitive electrochemical immunosensor via amide hydrolysis by DT-diaphorase combined with five redox-cycling reactions

Amide hydrolysis using enzyme labels, such as proteases, occurs at a slower rate than phosphoester and carboxyl ester hydrolysis. Thus, it is not very useful for obtaining high signal amplification in biosensors. However, amide hydrolysis is less sensitive to nonenzymatic spontaneous hydrolysis, allowing for lower background levels. Herein, we report that amide hydrolysis by DT-diaphorase (DT-D) occurs rapidly and that its combination with five redox-cycling reactions allows for the development of a highly sensitive electrochemical immunosensor. DT-D rapidly generates ortho-aminohydroxy-naphthalene (oAN) from its amide substrate via amide hydrolysis, which not even trypsin, a highly catalytic protease, can achieve. NADH, which is required for amide hydrolysis, advantageously acts as a reducing agent for rapid electrooxidation-based redox-cycling reactions. In the presence of oAN, DT-D, and NADH, two redox-cycling reactions rapidly occur. In the additional presence of an electron mediator, Ru(NH3)63+ [Ru(III)], three more redox-cycling reactions occur because Ru(III) reacts rapidly with oAN and DT-D. Although the O2-related redox-cycling reactions and redox reaction decrease electrochemical signals, this signal-decreasing effect is not significant in air-saturated solutions. The slow electrooxidation of NADH at an indium tin oxide electrode and sluggish reaction between NADH and Ru(III) allow for low electrochemical backgrounds. When the developed signal amplification scheme is tested for the sandwich-type electrochemical detection of parathyroid hormone (PTH), a detection limit of ∼1 pg/mL is obtained. The detection method is highly sensitive and can accurately measure PTH in serum samples.

Parahydrogen-Induced Hyperpolarization of Unsaturated Phosphoric Acid Derivatives

Parahydrogen-induced nuclear polarization offers a significant increase in the sensitivity of NMR spectroscopy to create new probes for medical diagnostics by magnetic resonance imaging. As precursors of the biocompatible hyperpolarized probes, unsaturated derivatives of phosphoric acid, propargyl and allyl phosphates, are proposed. The polarization transfer to 1H and 31P nuclei of the products of their hydrogenation by parahydrogen under the ALTADENA and PASADENA conditions, and by the PH-ECHO-INEPT+ pulse sequence of NMR spectroscopy, resulted in a very high signal amplification, which is among the largest for parahydrogen-induced nuclear polarization transfer to the 31P nucleus.

Intermolecular and Intramolecular Priming Co-directed Synergistic Multi-strand Displacement Amplification Empowers Ultrasensitive Determination of microRNAs.

The construction of isothermal nucleic acid amplification systems with extremely high signal amplification abilities is of great importance for biomarker detection and disease diagnosis. Herein, for the first time, we proposed an intermolecular and intramolecular priming co-directed synergistic multi-strand displacement amplification (SM-SDA) system for microRNA (miRNA) detection. Strategically, the SM-SDA system is made of a single multi-functionalized hairpin probe (MF-HP) that is engineered with a long stem and endowed with target complementation, configuration transformation, enzyme recognition, and signal reporting abilities. The presence of a specific target, the model of miRNA-21, to react with MF-HP would cause an intermolecular priming-directed strand replication in parallel with an intramolecular priming-directed strand replication. The co-directed priming pattern results in the occurrence of SM-SDA containing a target-induced cyclical strand displacement amplification (T-CSDA), a target analogue primer (TAP)-induced CSDA (TAP-CSDA), and a nicked trigger primer (NTP)-induced CSDA (NTP-CSDA). The resulting multiple circuits with a synergetic signal amplification capacity remarkably enhance the target miRNA response, which has not only improved the sensitivity for qualitative and quantitative detection of miRNA but also realized the analysis of target miRNA from real clinical samples. As a proof-of-concept study, this simple but attractive isothermal signal amplification system holds a great potential for molecular diagnosis of diseases and will stimulate interest, new ideas, and discoveries in this fascinating field.

Offset Compensation in Resistive Stretch Sensors Using Low-Frequency Feedback Topology

Respiration monitoring systems play an important role in healthcare and fitness. For this purpose, resistive stretch sensors are frequently used, which are cheap and simple in operation. However, they are not free from drawbacks. Varying offset due to patient movement, low signal amplitude, as well as susceptibility to interference, can all pose serious challenges. In this paper, a novel signal conditioning circuit for a resistive respiration sensor is proposed that alleviates some of the above problems. Namely, the proposed low-frequency feedback topology improves the dynamic range by offset compensation, sustaining a high signal amplification. Further advantages of the new configuration are the phase shift of 0.5 degrees in the band of interest and higher gain for the respiration signal than for the offset. The topology was proved to correctly represent signal amplitude changes, as well as to be able to sample human respiration in the home environment. However, the circuit shows some nonlinear behavior around resistance discontinuity points–settling time after body position change of the patient, which can be as long as 40 s. The circuit was tested both in bench tests and in the prototype of a respiratory polygraphy device during actual sleep apnea examinations. The results indicate that resistive stretch sensors, along with low-frequency feedback topology, are a promising development path for future respiration monitoring devices.

Dual Hydrazine-Equipped Turn-On Manganese-Based Probes for Magnetic Resonance Imaging of Liver Fibrogenesis.

Liver fibrogenesis is accompanied by upregulation of lysyl oxidase enzymes, which catalyze oxidation of lysine ε-amino groups on the extracellular matrix proteins to form the aldehyde containing amino acid allysine (LysAld). Here, we describe the design and synthesis of novel manganese-based MRI probes with high signal amplification for imaging liver fibrogenesis. Rational design of a series of stable hydrazine-equipped manganese MRI probes gives Mn-2CHyd with the highest affinity and turn-on relaxivity (4-fold) upon reaction with LysAld. A dynamic PET-MRI study using [52Mn]Mn-2CHyd showed low liver uptake of the probe in healthy mice. The ability of the probe to detect liver fibrogenesis was then demonstrated in vivo in CCl4-injured mice. This study enables further development and application of manganese-based hydrazine-equipped probes for imaging liver fibrogenesis.

Sensitive Affinity-Based Biosensor Using the Autocatalytic Activation of Trypsinogen Mutant by Trypsin with Low Self-activation.

Self-propagating autocatalytic reactions of proteases that can provide high signal amplification have not been applied to affinity-based biosensors owing to the limited number of fast autocatalytic proteolytic reactions available and the self-activation of protease proenzymes. Here, we report that a self-propagating autocatalytic reaction based on the autocatalytic activation of the trypsinogen mutant by trypsin facilitates high signal amplification and a low background level, resulting in a low detection limit for prostate-specific antigen (PSA). A commercially available trypsinogen mutant minimizes the self-activation of trypsinogen by trypsinogen. Trypsin, which is used as a catalytic label in a sandwich-type immunosensor, converts the trypsinogen mutant into trypsin; the generated trypsin then further converts the trypsinogen mutant into trypsin. The autocatalytically produced trypsin proteolytically cleaves the peptide bond of a trypsin substrate, resulting in the liberation of electrochemically active 4-aminophenol (AP). The electrochemical oxidation of AP at a modified indium tin oxide (ITO) electrode induces electrochemical-chemical redox cycling involving the ITO electrode, AP, and a reductant. The triple combination of autocatalytic activation, proteolytic cleavage, and redox cycling results in a high electrochemical signal level. The detection limit for PSA obtained using a trypsin label and trypsinogen (∼7 pg/mL) is lower than that obtained using a trypsin label alone (∼100 pg/mL). This study demonstrated that autocatalytically activating a proenzyme is a very useful method for highly amplifying signals.

Self-Customized Multichannel Exponential Amplifications Regulate Powered Monitoring of Terminal Deoxynucleotidyl Transferase Activity.

The discovery and function analysis of terminal deoxynucleotidyl transferase (TdT) add a new dimension to the understanding of leukemia mechanisms and stimulate the development of new analytical tools for leukemia diagnosis. Herein, taking advantage of the inherent property of TdT for performing DNA synthesis using only single-stranded DNA as the nucleic acid substrate, we developed a self-customized multichannel exponential amplification (SMEA) system for the fluorescent sensing of TdT activity. The SMEA design employs an intermolecular DNA interaction made of a nicking site-incorporated elongation primer (EP) and a nicking site-incorporated poly-thymine tailed molecular beacon (Poly-T-MB). The absence of TdT is unable to bridge the relationship between EP and Poly-T-MB, ensuring the SMEA has an ultralow background. The presence of TdT, however, leads to the elongation of EP to poly-adenine tailed EP (Poly-A-EP) under a dATP pool responsible for further hybridization with numerous Poly-T-MB. With the aid of polymerase and nickase to react with the hybridization product of Poly-A-EP/(Poly-T-MB)n, it can cause bidirectional strand nicking, polymerization, and displacement in many cycles and channels. In this case, the SMEA is found to be associated with the configuration transformation and splitting of all Poly-T-MBs for a significant fluorescence enhancement. Depending on this high target signal amplification and strong background inhibition abilities, the SMEA sensing system is powerful for the qualitative and quantitative determination of TdT activity, showing that it has great promise for biomedical study and disease diagnosis.

Coulometric ion sensing with Li+-selective LiMn2O4 electrodes

A coulometric signal readout method, which was originally developed for solid-contact ion-selective electrodes, was investigated in this work using LiMn2O4 (LMO) as a combined ion-recognition and signal-transduction layer. The redox process of LMO, which is associated with reversible intercalation/expulsion of Li+ ions, allowed coulometric sensing of Li+ ions in aqueous solutions. On increasing the active area (mass loading) of LMO, the coulometric signal increased for a given change in Li+ ion activity. The excellent redox reversibility of LMO and its relatively low resistance were instrumental in achieving a high signal amplification together with a relatively fast response. Coating the LMO layer with a conventional Li+-selective plasticized PVC membrane was found to dramatically lower the coulometric response. Hence, the application of LMO as a combined Li+-selective electrode material and ion-to-electron transducer was found to be highly compatible with the coulometric signal readout method, especially for detecting small Li+ activity changes at high Li+ concentrations.

Hollow covalent organic framework-sheltering CRISPR/Cas12a as an in-vivo nanosensor for ATP imaging

In addition to applications in genome editing, clustered regularly interspaced short palindromic repeats (CRISPR) have recently been engineered for medical diagnostics based on their trans-cleavage activity owing to their high base resolution and isothermal signal amplification. However, trans-cleavage activity is too fragile to be applied in vivo. Herein, we introduce a hollow covalent organic framework (COF)-sheltering CRISPR/aptamer-based sensor (h-CCS) for ATP imaging in living animals. The CRISPR/aptamer-based complex is comprised of the CRISPR-Cas12a system, fluorophore quencher-labeled single-stranded DNA substrate (ssDNA-FQ), and a DNA activator that pre-hybridizes with ATP aptamer to prevent the trans-cleavage activity of the Cas12a system in the absence of ATP. After being encapsulated in a hollow COF, the constructed nanoreactor is highly robust and can be lit up by ATP for in vivo imaging. Considering the unique properties of h-CCS, this strategy offers great potential to broaden applications of not only CRISPR-Cas systems but also other proteins in porous matrixes for clinical diagnostics, medical research, and biomimetic nanodevices.