Signal Amplification For Ultrasensitive Detection Research Articles

Portable SERS biosensor based on aptamer-assisted catalytic hairpin assembly signal amplification for ultrasensitive detection of Staphylococcus aureus

Bacteria infections pose a serious threat to public health, and it is urgent to develop facile and accurate detection methods. To meet the important need, a potable and high-sensitive surface enhanced Raman scattering (SERS) biosensor based on aptamer recognition and catalytic hairpin assembly (CHA) signal amplification was proposed for point-of-care detection of Staphylococcus aureus (S. aureus). The SERS biosensor contains three parts: recognition probes, SERS sensing chip, and SERS tags. The feasibility of the strategy was verified by gel electrophoresis, and the one-step test route was optimized. The bacteria SERS biosensor has a good linear relationship ranging from 10 to 107 CFU·mL-1 with high sensitivity low to 5 CFU·mL-1, and shows excellent specificity, uniformity, and repeatability on S. aureus identification and enumeration, which can distinguish S. aureus from other bacteria. The SERS biosensor shows a good recovery rate (95.73% to 109.65%) for testing S. aureus spiked in milk, and has good practicability for detecting S. aureus infected mouse wound, which provides a facile and reliable approach for detection of trace bacteria in the real samples.

Ultrathin metal-organic framework nanosheets (Cu-TCPP) assembled micro flower combined with endonuclease signal amplification for ultrasensitive detection

Metal-organic framework play an important role in developing novel detection techniques. Herein, an ultrathin two-dimensional (2D) metal-organic framework nanosheet (Cu-TCPP) assembled microflower was successfully synthesized and applied for ultrasensitive detection. The high detection performance of Cu-TCPP microflower was attributed to high-capacity aptamer adsorption and specific desorption resulting from numerous approachable active sites, and its ultrathin nanosheets microflower-like structure. Moreover, the Cu-TCPP microflower had overcame the disadvantages of 2D MOF nanosheets which tended to agglomerate. Moreover, it exhibited excellent fluorescence quenching performance as a donor for fluorescence resonance energy transfer. Acetamiprid, a new chloronicotinic neurotoxic insecticide was used as the model analyte. The ultrasensitive sensor was developed by combining the Cu-TCPP microflower with deoxyribonuclease Ⅰ signal enhanced fluorescence. When acetamiprid was present, the fluorescent labeled aptamer probe was selectively bound to acetamiprid and desorbed from the Cu-TCPP microflower, resulting in fluorescence recovery. Subsequently, DNase Ⅰ was used to cleave the DNA strands of the aptamer probe, and acetamiprid was released to participate in a new cycle. The detection limit of the assay was as low as 5.56 pg/mL with a linear range 50.00 pg/mL∼5.00 μg/mL, and it was a universal platform to detect other analytes.

“Two-step” signal amplification for ultrasensitive detection of dopamine in human serum sample using Ti3C2Tx-MXene

Two-dimensional transition-metal (Ti3C2Tx-MXene) carbide layered materials with versatile surface functional group have attracted huge interest in electrochemical sensing due to their excellent metallic conductivity, outstanding electrical and mechanical properties, high volumetric capacity, and flexibility to construct highly sensitive biosensors. Herein, we present a ultrasensitive molecular architecture for neuromolecules dopamine sensing using Ti3C2Tx-MXene multilayered materials. It was prepared through in situ polymerization of 4-APBA (4-aminophenyl boronic acid) with a scalable “two-step” approach. Additionally, its on-site application was realized by fabricating a label-free screen-printed carbon (SPC) electrode for electrochemical sensing of dopamine levels in buffer and human serum samples at the point-of-care testing (POCT). Importantly, the high specificity of Ti3C2Tx-MXene/4-APBA modification for SPC-electrode electrochemical sensing could be easily implemented with great signal drifting. As a proof of concept, this novel electrochemical sensor was found to be logically inexpensive and straightforward. It could selectively measure dopamine levels with a limit of detection (LOD) of 1.3 nM in PBS (pH 7.2) and, 1.9 nM in the human serum sample. It had a wide range of linear detection of dopamine concentrations of 40 to 500 nM, with a sensitivity of 0.0134 µA nM−1. This work paves a new path for 2D Ti3C2Tx-MXene/4-APBA modification for biomedical and biosensing applications in dopamine detection at nanomolar concentrations with high specificity, selectivity, sensitivity, stability, and reproducibility.

MOF-based nanocomposites coupled with RecJf exonuclease-assisted target recycling amplification: Dual signal amplification for ultrasensitive detection of vanillin

A “signal off” electrochemical aptasensor based on metal-organic framework (MOF) - based nanocomposites and RecJf exonuclease-assisted target recycling was constructed for ultrasensitive detection of vanillin (VAN). In this work, benefited from the high catalytic activity of the synthesized gold nanoparticle/iron-based MOF/polyetherimide-reduced graphene oxide (AuNPs/NH2-MOF-235(Fe)/PEI-rGO) and the AuNPs/Ce-MOF nanocomposites toward thionine (Thi), the electrochemical signal was amplified. And the target recycling assisted by RecJf exonuclease were triggered with the presence of VAN, which induced the strongly reduced electrochemical signal. On account of dual amplification strategies, the performance of the aptasensor was significantly improved to achieve ultrasensitive detection of VAN. Under optimal conditions, the linear relationship was fitted for VAN with a linear range of 2 pM - 0.2 mM and the detection limit of 1.14 pM (S/N = 3). Furthermore, this assay for VAN has excellent specificity, long-term stability, and satisfactory results of the detection in the real samples. In summary, the aptasensor could be further used for the specific quantitative determination of other targets in foods.

Reusable electrochemiluminescence biosensor based on tetrahedral DNA signal amplification for ultrasensitive detection of microRNAs.

A novel and reusable electrochemiluminescence biosensor was developed based on tetrahedral DNA (TDN) signal amplification for ultrasensitive detection of miRNA-27a. The flowered nickel-iron layered double hydroxide@AuNPs (NiFe-LDH@AuNPs) composites increase the amount of hairpin DNA fixed on the electrode. When miRNA is present, TDN-Ru(bpy)32+ acts as an ECL probe, forming a stable sandwich structure with miRNA-27a and hairpin DNA through base complementation pairing, thus achieving miRNA detection. This biosensor has the characteristics of high sensitivity, excellent selectivity, and good reproducibility.

Aptamer-based chemiluminescent optical fiber immunosensor with enhanced signal amplification for ultrasensitive detection of tumor biomarkers

With the growing demand for early diagnosis of malignant tumor, ultrasensitive detection of cancer-related biomolecules with convenient operation is of great significance for timely treatment. In this study, we develop a robust and efficient analytical system, the aptamer-based chemiluminescent optical fiber immunosensor (aptamer-COFIS), for ultrasensitive detection of biomarkers. In the aptamer-COFIS system, target aptamer-primer probe is used as the bridge to combine antibody-antigen immunoreaction on the fiber surface with chemiluminescent signal amplification. The antibody-antigen (target)-aptamer sandwich-like reaction is coupled with rolling cycle amplification (RCA), which is capable to produce hundreds of binding sites to connect with the streptavidin-biotinylated horseradish peroxidase nanocomposites for signal amplification. Using prostate specific antigen (PSA), the most reliable and specific biomarker of prostate cancer, as the test sample, we show that the proposed strategy can greatly improve the sensor response with a wide linear range of 0.01–1000 pg/mL and an extremely low limit-of-detection of 3.2 fg/mL. Our results indicate that the proposed method presents high selectivity and good accuracy for ultrasensitive sensing biomarkers in complex clinical samples. Moreover, with additional advantages by using optical fiber as the ideal carrier of biorecognition elements and the transducer of chemiluminescent signal, the aptamer-COFIS system with a low-cost compact design is convenient for operation, making it suitable for cost-effective on-site and real time analysis of various biomarkers in early diagnosis of diseases.

Intracellular Signal Amplification for Ultrasensitive Detection and Imaging: Progress, Challenges, and Opportunities

AbstractDisease signaling molecules and biomarkers are important in biosensing and biomedicine, and their ultra‐sensitive detection and imaging are critical in medical diagnosis. However, most biomarkers have low levels of expression in cells and are easily degraded; therefore, they cannot be accurately detected in real time by conventional amplification methods, which are typically confined to cell lysates. To achieve highly sensitive detection of intracellular low‐abundance molecules, extensive efforts have been devoted to the development of in situ signal amplification techniques. This review tracks the development of recent advances in in situ signal amplification strategies, and systematically summarizes the advantages and disadvantages of enzymatic and non‐enzymatic amplification techniques for detecting and tracing intracellular targets. Further, perspectives, challenges, and opportunities for intracellular signal amplification are discussed to promote its adoption for in situ signal amplification in clinical settings and to provide new insights into signaling molecules and biomarkers in cellular functions and associated diseases.

Aptamer-Based Chemiluminescent Optical Fiber Immunosensor with Enhanced Signal Amplification for Ultrasensitive Detection of Tumor Biomarkers

Aptamer-Based Chemiluminescent Optical Fiber Immunosensor with Enhanced Signal Amplification for Ultrasensitive Detection of Tumor Biomarkers

Isothermal cross-boosting extension-nicking reaction mediated exponential signal amplification for ultrasensitive detection of polynucleotide kinase.

A novel nucleic acid-based isothermal signal amplification strategy, named cross-boosting extension-nicking reaction (CBENR) is developed and successfully used for rapid and ultrasensitive detection of polynucleotide kinase (PNK) activity. Only two simple oligonucleotides (recognition substrate (RS) and TaqMan probe) are applied to construct the PNK-sensing platform. In the presence of PNK, the 3'-phosphate end of RS will be converted to the 3'-hydroxyl one, and then extended to a long poly-adenine (poly-A) sequence under the catalysis of terminal deoxynucleotidyl transferase (TdT). The poly-A sequence provides multiple binding sites for the TaqMan probe to form multiple DNA duplexes. Subsequently, ribonuclease HII (RNase HII) cuts the TaqMan probe into two parts at the pre-set uracil site, generating a fluorescence signal and providing new substrates for TdT elongation. The TdT-catalyzed substrate extension and RNase HII-catalyzed probe nicking are boosted by each other, resulting in persistent enlargement of these two reactions and thus giving ultrahigh signal amplification efficiency. Utilizing the CBENR-based PNK sensor, ultrasensitive detection of PNK activity was achieved with a detection limit as low as 3.0 × 10-6 U mL-1. Quantification of endogenous PNK activity at the single-cell level and the screening/evaluation of PNK inhibitors were also achieved.

Ultrasensitive detection of NDM-1 resistant bacteria based on signal amplification with sandwich-type LNA electrochemical biochips

New Delhi metallo-β-lactamase-1 (NDM-1) is an identified metallo-β-lactamase that confers resistance to carbapenems and all other β-lactam antibiotics, with the exception of aztreonam. Herein, a novel electrochemical biochip was constructed based on a zinc oxide (ZnO) and reduced graphene oxide (rGO) complex (ZnO@rGO) supported by Au nanocages (Au NCs) for signal amplification for ultrasensitive detection of NDM-1 in early clinical diagnosis. A Au NC and a sulfhydryl (SH) modified locked nucleic acid-1 (Au NC@LNA-1) probe was synthesized with stable Au-S bonds to on the biochip surface. In hybridization solution, Au NCs and LNA-2 probes modified with SH groups formed stable Au NC@LNA-2 complexes. Au NC@LNA-1 and Au NC@LNA-2 were linked with the NDM-1 to form a Au NC@LNA-1/NDM-1/Au NC@LNA-2 “sandwich-like” complex through complementary base pairing, and this complex can bind additional copies of the target sequence to further amplify it’s the detection signal. Under optimized conditions, the system showed a linear range from 1 pg L−1 to 100 μg L−1, and the detection limit was 0.042 pg L−1. The time of biochip probe-modification was 45 min, incubation was 30 min, and electrochemical scanning detection was 1 min. In addition, the results indicate that the specific LNA probe identified single base mismatches and exhibited affinity for the target DNA molecule, resulting in the direct detection of the NDM-1 in complex clinical bacteria samples without PCR amplification. In conclusion, this novel electrochemical biochip method allows for the rapid, specific, and sensitive detection of the NDM-1, indicating its potential application in disease diagnostics.

Aptamer recognition and proximity-induced entropy-driven circuit for enzyme-free and rapid amplified detection of platelet-derived growth factor-BB

Platelet-derived growth factor-BB (PDGF-BB) is currently used as a biomarker protein for cancer early diagnosis and clinical treatment. Herein, we reported a robust and enzyme-free strategy based on aptamer recognition and proximity-induced entropy-driven circuits (AR-PEDC) for homogeneous and rapid detection of platelet-derived growth factor BB (PDGF-BB) without any washing steps or thermocycling. The proximity probes specifically recognize target protein to form the completed trigger (CT). Then, the CT reacts with three-strand complex to form intermediate, which subsequently binds to fuel strand to release reporter strand, assistant strand and the CT. The revised proximity probes exhibit significantly improved signal-to-background ratio and faster association rate. Moreover, target protein/proximity probes interaction can specifically initiate entropy-driven circuits, thus providing immense signal amplification for ultrasensitive detection of PDGF-BB with low detection limit of 9.6 pM. The practical ability of the developed strategy is demonstrated by detection of PDGF-BB in human serum with satisfactory results. In addition, this method is flexible and can be conveniently extended to a variety of targets by simply substituting the target specific sequence. Thus, this strategy presents a rapid, low background and versatile amplification mechanism for the detection of protein biomarkers and offers a promising alternative platform for clinical diagnosis.

Target-assisted FRET signal amplification for ultrasensitive detection of microRNA

MicroRNA (miRNA) is a type of noncoding RNA and plays a crucial role in gene expression regulation. Sensitive identification and detection of miRNA can offer vast information for transcriptome analysis and cancer studies. Conventional PCR and molecular hybridization techniques suffer from low specificity when used for miRNA detection due to the short nucleotide length of miRNA. The extremely low abundance of miRNA in real biological samples also sets higher demands for the sensitivity of detection methods. A novel method based on target-assisted fluorescence resonance energy transfer (FRET) signal amplification was proposed for the simple and ultrasensitive detection of miRNA. A hairpin fluorescence DNA probe could hybridize with target miRNA to expose the hybridization site for the primer. After being duplexed with the nanogold-labeled primer, the fluorescent dye in the DNA probe was quenched via the FRET mechanism. In the presence of a DNA polymerase, primer-activated strand displacement released the miRNA, and then the miRNA strand could recognize and function with another DNA probe. The recycling of target miRNA and a high quenching efficiency of nanogold greatly improved the sensitivity. The detection limit of the method (1.5 fM) was lower than that of the reported strategies using a target cycling reaction, allowing trace detection of miRNA in real samples. By making full use of miRNA sequences, the method could also recognize single-base mismatches and distinguish homologous miRNAs.

A novel electrochemical immunosensor based on Au nanoparticles and horseradish peroxidase signal amplification for ultrasensitive detection of α-fetoprotein.

An electrochemical double-layer Au nanoparticle membrane immunosensor was developed using an electrochemical biosensing signal amplification system with Au nanoparticles, thionine, chitosan, and horseradish peroxidase, which was fabricated using double self-adsorption of Au nanoparticle sol followed by anti-α-fetoprotein Balb/c mouse monoclonal antibody adsorption. The AuNPs sol was characterized by spectrum scanning and transmission electron microscopy. The immunosensor was characterized by atomic force microscopy, cyclic voltammetry, and alternating-current impedance during each stage of adsorption and assembly. The amperometric I-t curve method was used to measure α-fetoprotein (AFP) diluted in phosphate buffered saline. The result indicated a wide linear range, and the change rate of steady-current before and after immune response had linear correlation within the range 0.1-104pg/mL AFP. The current change rate equation was △I = 5.82334 lgC + 37.01195 (R2 = 0.9922). The lowest limit of detection was 0.03pg/mL (S/N= 3), and the reproducibility of the sensor was good. Additionally, the sensor could be stably stored above phosphate buffered saline at 4°C for more than 24days. More importantly, the sensor is label-free, reagentless and low fouling, making it capable of assaying AFP in real serum samples without suffering from significant interference or biofouling.

MRNA-Initiated, Three-Dimensional DNA Amplifier Able to Function inside Living Cells.

DNA molecular machines show great promise in fields such as biomarker discovery and biological activity regulation, but operating DNA machines with specific functions within living systems remains extremely challenging. Although DNA machines have been engineered with exact molecular-level specifications, some intrinsic imperfections such as poor cell permeation and fragility in complex cytoplasmic milieu persist due to the well-established character of nucleic acid molecules. To circumvent these problems, we herein report a molecularly engineered, entropy-driven three-dimensional DNA amplifier (EDTD) that can operate inside living cells in response to a specific mRNA target. In particular, mRNA target/EDTD interaction can specifically initiate an autonomous DNA circuit inside living cells owing to the exclusive entropy-driven force, thus providing enormous signal amplification for ultrasensitive detection of the mRNA. Moreover, owing to molecular engineering of a unique DNA tetrahedral framework into the DNA amplifier, EDTD exhibits significantly enhanced biostability and cellular uptake efficiency, which are prerequisites for DNA machines used for in vivo applications. This programmable DNA machine presents a simple and modular amplification mechanism for the detection of intracellular biomarkers. Moreover, this study provides a potentially valuable molecular tool for understanding the chemistry of cellular systems and offers a design blueprint for further expansion of DNA nanotechnology in living systems.

Excision Repair-Initiated Enzyme-Assisted Bicyclic Cascade Signal Amplification for Ultrasensitive Detection of Uracil-DNA Glycosylase

Uracil-DNA glycosylase (UDG) is an important base excision repair (BER) enzyme responsible for the repair of uracil-induced DNA lesion and the maintenance of genomic integrity, while the aberrant expression of UDG is associated with a variety of cancers. Thus, the accurate detection of UDG activity is essential to biomedical research and clinical diagnosis. Here, we develop a fluorescent method for ultrasensitive detection of UDG activity using excision repair-initiated enzyme-assisted bicyclic cascade signal amplification. This assay involves (1) UDG-actuated uracil-excision repair, (2) excision repair-initiated nicking enzyme-mediated isothermal exponential amplification, (3) ribonuclease H (RNase H)-induced hydrolysis of signal probes for generating fluorescence signal. The presence of UDG enables the removal of uracil from U·A pairs and generates an apurinic/apyrimidinic (AP) site. Endonuclease IV (Endo IV) subsequently cleaves the AP site, resulting in the break of DNA substrate. The cleaved DNA substrate functions as both a primer and a template to initiate isothermal exponential amplification, producing a large number of triggers. The resultant trigger may selectively hybridize with the signal probe which is modified with FAM and BHQ1, forming a RNA-DNA heterogeneous duplex. The subsequent hydrolysis of RNA-DNA duplex by RNase H leads to the generation of fluorescence signal. This assay exhibits ultrahigh sensitivity with a detection limit of 0.0001 U/mL, and it can even measure UDG activity at the single-cell level. Moreover, this method can be applied for the measurement of kinetic parameters and the screening of inhibitors, thereby providing a powerful tool for DNA repair enzyme-related biomedical research and clinical diagnosis.

Gold Nanoparticles and Cleavage-Based Dual Signal Amplification for Ultrasensitive Detection of Silver Ions

Silver ion (Ag(+)) is a highly toxic heavy metal ion to fungi, viruses, bacteria, and animals. Therefore, Ag(+) monitoring in water or food resources has become extraordinarily important within the scope of human health. Here, we report a gold nanoparticles and enzyme cleavage-based dual signal amplification strategy for ultrasensitive detection of Ag(+) using electrochemical techniques. This sensing platform for Ag(+) has an extremely low detection limit of 470 fM, which also has satisfactory selectivity. Thus, it can be directly used in drinking water and lake water samples. Moreover, the strategy proposed in this work may have potential to be further developed as a generalized platform for the detection of other analytes by designing new DNA sequences for specific recognition.

Electrically contacted enzyme based on dual hairpin DNA structure and its application for amplified detection of Hg2+

In the present study, based on a dual hairpin DNA structure, a novel system of electrically contacted enzyme and its signal amplification for ultrasensitive detection of Hg2+ was demonstrated. In the presence of Hg2+, with the interaction of thymine–Hg2+–thymine (T–Hg2+–T), DNA sequence dully labeled with ferrocene (Fc) at 5′ end and horseradish peroxidase (HRP) at 3′ end, hybridized to the capture probe and formed the dual hairpin structure on the electrode. Fc unit acts as a relay that electrically contacts HRP with the electrode and activates the bioelectrocatalyzed reduction of H2O2. And based on the bioelectrocatalyzed signal amplification of the presented system, Hg2+ could be quantitatively detected in the range of 10−10–10−6M with a low detection limit of 52pM. And it also demonstrated excellent selectivity against other interferential metal ions.

The strategy of signal amplification for ultrasensitive detection of hIgE based on aptamer-modified poly(di-acetylene) supramolecules

Herein, we demonstrate three strategies of signal amplification for ultrasensitive detection of human immunoglobulin E (hIgE) based on poly(di-acetylene) supramolecules. To fabricate the ultrasensitive PDA biosensor, ethylenediamine as an interlinker and aptamer as a receptor were introduced into the chip fabrication process. Using the prepared PDA liposome biosensor, the hIgE could be detected up to below 1.0 ng/ml by a primary response. In order to accomplish more ultrasensitive detection of protein on a PDA biosensor, polyclonal hIgE antibody was employed as an external mechanical force for the inducement of a secondary response. As a result, a PDA liposome biosensor sensitivity as high as 0.01 ng/ml for the target hIgE was obtained, with a sensitivity which is one hundred times of that of the method without signal amplification. These results indicate that the proposed strategies were capable of ultrasensitive quantitative and qualitative analyses of biomolecules without non-specific binding of non-target proteins.

Hairpin DNA Switch for Ultrasensitive Spectrophotometric Detection of DNA Hybridization Based on Gold Nanoparticles and Enzyme Signal Amplification

A novel DNA detection platform based on a hairpin DNA switch, nanoparticles, and enzyme signal amplification for ultrasensitive detection of DNA hybridization has been developed in this work. In this DNA assay, a "stem-loop" DNA probe dually labeled with a thiol at its 5' end and a biotin at its 3' end, respectively, was used. This probe was immobilized on the gold nanoparticles (AuNPs) anchored by a protein, gamma-globulin, on a 96-well microplate. In the absence of target DNA, the immobilized probe with the stem-loop structure shields the biotin from being approached by a bulky horseradish peroxidase linked streptavidin (streptavidin-HRP) conjugate due to the steric hindrance. However, in the presence of target DNA, the hybridization between the hairpin DNA probe and the target DNA causes significant conformational change of the probe, which forces biotin away from the surface of AuNPs. As a result, the biotin becomes accessible by the streptavidin-HRP, and the target hybridization event can be sensitively detected via the HRP catalyzed substrate 3,3',5,5'-tetramethylbenzidine using a spectrophometric method. Some experimental parameters governing the performance of the assay have been optimized. At optimal conditions, this DNA assay can detect DNA at the concentration of femtomolar level by means of a signal amplification strategy based on the combination of enzymes and nanoparticles. This approach also has shown excellent specificity to distinguish single-base mismatches of DNA targets because of the intrinsic high selectivity of the hairpin DNA probe.