Jinming Kong

Ultrasensitive electrochemical DNA biosensor by exploiting hematin as efficient biomimetic catalyst toward in situ metallization

In this work, we presented a novel signal amplification approach to construct an electrochemical DNA biosensor for the ultrasensitive determination of sequence-specific DNA by exploiting hematin as biomimetic catalyst toward in situ metallization. Briefly, thiolated peptide nucleic acid (PNA) probes were firstly immobilized onto gold electrode through the formation of self-assembled monolayer (SAM) and then hybridization was accomplished in the ensuing step. After that, hematin molecules were introduced to the hybridized PNA/DNA heteroduplexes by employing phosphate-zirconium-carboxylate coordination chemistry. Next, the attached hematin molecules acted as catalyst in accelerating the reduction of silver ions in the presence of catechol, leading to the in situ deposition of silver particles onto the electrode. Finally, the deposited silver particles were electrochemically stripped into KCl solution and measured by square wave voltammetry (SWV). Under optimal conditions, the hematin-based electrochemical DNA biosensor presented a good linear relationship between the stripping peak currents and logarithm of single-stranded DNA (ssDNA) concentrations in the range from 0.1 fM to 0.1 nM with a low detection limit of 62.41 aM, and it rendered satisfactory analytical performance for the determination of ssDNA in serum samples. Furthermore, it exhibited good reproducibility and stability, meanwhile, it also showed excellent specificity toward single-nucleotide polymorphism (SNP). Therefore, the hematin-based signal amplification approach has great potential in clinical applications and is also suitable for quantification of biomarkers at ultralow level.

Sensitive and selective colorimetric assay of alkaline phosphatase activity with Cu(II)-phenanthroline complex

Alkaline phosphatase (ALP) plays a vital role in dephosphorylation- and phosphorylation-related cellular regulation and signaling processes. Accordingly, the development of efficient methods for ALP activity assay is of significant importance in clinical diagnosis. In this work, a simple and practical method is reported for the first time for the sensitive and selective colorimetric assay of ALP activity by exploiting a water-soluble Cu(II)-phenanthroline complex as the probe, on the basis of the distinctive metal-to-ligand charge-transfer (MLCT) properties. This method is simply built on a two-step chromogenic reaction: the enzymatic hydrolysis of the substrate ascorbic acid 2-phosphate to ascorbic acid (AA), followed by the reduction of the colorimetric probe Cu(BPDS)22- (BPDS=bathophenanthroline disulfonate) by AA to its cuprous form. The latter process triggers a turn-on spectral absorption at 424nm and a striking color change of the solution from colorless to blackish-green. Needless of complicated protocols and instrumentation, this method allows a sensitive readout of ALP activity within a wide linear range of 0-200mUmL-1, with a detection limit down to 1.25mUmL-1. Results also reveal that it is highly selective and holds great potential in ALP inhibitor efficiency evaluation. In addition, quantitative analysis of ALP activity in spiked serum samples has been realized successfully in the linear range of 0-200mUmL-1, with a detection limit of 1.75mUmL-1. Advantages of simplicity, wide linear range, high sensitivity and selectivity, low cost, and little background interference render this method great potential in practical applications.

Turn-On Colorimetric Platform for Dual Activity Detection of Acid and Alkaline Phosphatase in Human Whole Blood.

The activity detection of acid phosphatase (ACP) and alkaline phosphatase (ALP) is of great importance to the diagnosis and prognosis of related diseases. In this work, we report for the first time a turn-on colorimetric platform for the activity detection of ACP and ALP, by exploiting Cu(BCDS)22- (BCDS=bathocuproinedisulfonate) as the probe. The presence of ACP or ALP dephosphorylates the substrate ascorbic acid 2-phosphate to produce ascorbic acid, which then reduces Cu(BCDS)22- into Cu(BCDS)23- , leading to a turn-on spectral absorption at 484 nm and a dramatic color change of the solution from colorless to orange-red. The underlying metal-to-ligand charge-transfer mechanism has been demonstrated by quantum mechanical computations. This platform allows a rapid, sensitive readout of ACP and ALP activities within the dynamic range from 0 to 220 mU ml-1 . In addition, it is highly immune to false-positive results and also highly selective. More importantly, it is applicable in the presence of human serum and even whole blood samples. These results demonstrate that our platform holds great potential in clinical practices and in the point-of-care analysis.

Electrochemically Mediated Surface-Initiated de Novo Growth of Polymers for Amplified Electrochemical Detection of DNA

The development of convenient and efficient strategies without involving any complex nanomaterials or enzymes for signal amplification is of great importance in bioanalytical applications. In this work, we report the use of electrochemically mediated surface-initiated atom transfer radical polymerization (SI-eATRP) as a novel amplification strategy based on the de novo growth of polymers (dnGOPs) for the electrochemical detection of DNA. Specifically, the capture of target DNA (tDNA) by the immobilized peptide nucleic acid (PNA) probes provides a high density of phosphate groups for the subsequent attachment of ATRP initiators onto the electrode surface by means of the phosphate-Zr4+-carboxylate chemistry, followed by the de novo growth of electroactive polymer via the SI-eATRP. De novo growth of long polymeric chains enables the labeling of numerous electroactive probes, which in turn greatly improves the electrochemical response. Moreover, it circumvents the slow kinetics and poor coupling efficiency encountered when nanomaterials or preformed polymers are used and features sufficient flexibility and simplicity in controlling the degree of signal amplification. Under optimal conditions, it allows a highly sensitive and selective detection of tDNA within a broad linear range from 0.1 fM to 0.1 nM (R2 = 0.996), with the detection limit down to 0.072 fM. Compared with the unamplified method, more than 1.2 × 106-fold sensitivity improvement in DNA detection can be achieved. By virtue of its simplicity, high efficiency, and cost-effectiveness, the proposed dnGOPs-based signal amplification strategy holds great potential in bioanalytical applications for the sensitive detection of biological molecules.

A signal-on electrochemical DNA biosensor based on potential-assisted Cu(I)-catalyzed azide-alkyne cycloaddition mediated labeling of hairpin-like oligonucleotide with electroactive probe

A novel electrochemical biosensor was developed for the signal-on detection of sequence-specific DNA by exploiting potential-assisted Cu(I)-catalyzed azide-alkyne cycloaddition (φCuAAC) as an efficient approach for the labeling of hairpin-like oligonucleotide (hairpin) with electroactive probe. The hairpins, dually labeled with thiol and azide at either terminal, were firstly self-assembled on gold electrode and served as the capture probes for the specific recognition of target DNA. Upon hybridization with target DNA, the surface-confined hairpins were unfolded, liberating the azide-containing terminals away from electrode surface. Subsequently, the unfolded hairpins were conveniently and efficiently labeled with ethynylferrocene (EFC) via the φCuAAC. The quantitatively labeled EFC was finally measured via differential pulse voltammetry (DPV) for the signal-on electrochemical detection of sequence-specific DNA. The biosensor presented a good linear response over the range from 1pM to 1nM with a detection limit of 0.62pM. Results also revealed that it was highly specific and held a good detection capability in serum samples. Furthermore, the ability to chemoselectively label hairpin-like oligonucleotide with signal reporter by electrical addressing, together with the simplicity and efficiency of the φCuAAC, makes it compatible with microfluidic devices and microelectrode arrays to achieve the miniaturized and multiplexed detections.

Detection of sequence-specific DNA with a morpholino-functionalized silicon chip

In this work, an efficient method for the sequence-specific detection of DNA based on a morpholino-functionalized silicon chip platform has been proposed. Briefly, morpholino was first immobilized on the surface of a silicon chip using 3-aminopropyltriethoxysilane (APTES) as the silane coupling agent and 1,4-phenylenediisothiocyanate (PDITC) as the cross-linker and then hybridized with DNA in the ensuing step. The fluorescence label was introduced by strongly binding Rhodamine B, which contains a terminal carboxylic group, with DNA by means of phosphate–zirconium–carboxylate coordination reaction. X-ray photoelectron spectroscopy (XPS) was used to characterize the silicon surface. Under optimal conditions, the morpholino-functionalized silicon chip presented a great linear relationship between the fluorescence intensity and the logarithm of target DNA concentrations in the range from 1 pM to 1 nM with a detection limit of 4.52 pM. Furthermore, fully complementary versus single-base mismatched, three-base mismatched and non-complementary DNA could be effectively identified. The chip showed excellent stability because it could be reused for another hybridization experiment after denaturing the morpholino–complementary DNA duplex. In addition, the chip rendered satisfactory analytical performance for the detection of DNA in serum samples, thus exhibiting practical significance. Morpholino-functionalized silicon chips display good sensitivity and selectivity for the detection of DNA and promising applications in single-nucleotide polymorphisms (SNPs).

Fluorescence quenching based alkaline phosphatase activity detection

Simple and fast detection of alkaline phosphatase (ALP) activity is of great importance for diagnostic and analytical applications. In this work, we report a turn-off approach for the real-time detection of ALP activity on the basis of the charge transfer induced fluorescence quenching of the Cu(BCDS)22- (BCDS = bathocuproine disulfonate) probe. Initially, ALP can enzymatically hydrolyze the substrate ascorbic acid 2-phosphate to release ascorbic acid (AA). Subsequently, the AA-mediated reduction of the Cu(BCDS)22- probe, which displays an intense photoluminescence band at the wavelength of 402nm, leads to the static quenching of fluorescence of the probe as a result of charge transfer. The underlying mechanism of the fluorescence quenching was demonstrated by quantum mechanical calculations. The Cu(BCDS)22- probe features a large Stokes shift (86nm) and is highly immune to photo bleaching. In addition, this approach is free of elaborately designed fluorescent probes and allows the detection of ALP activity in a real-time manner. Under optimal conditions, it provides a fast and sensitive detection of ALP activity within the dynamic range of 0-220mUmL-1, with a detection limit down to 0.27mUmL-1. Results demonstrate that it is highly selective, and applicable to the screening of ALP inhibitors in drug discovery. More importantly, it shows a good analytical performance for the direct detection of the endogenous ALP levels of undiluted human serum and even whole blood samples. Therefore, the proposed charge transfer based approach has great potential in diagnostic and analytical applications.

Electrochemically mediated polymerization for highly sensitive detection of protein kinase activity

Protein kinases play a pivotal role in cellular regulation and signal transduction, the detection of protein kinase activity and inhibition is therefore of great importance to clinical diagnosis and drug discovery. In this work, a novel electrochemical platform using the electrochemically mediated polymerization as an efficient and cost-effective signal amplification strategy is described for the highly sensitive detection of protein kinase activity. This platform involves 1) the phosphorylation of substrate peptide by protein kinase, 2) the attachment of alkyl halide to the phosphorylated sites via the carboxylate-Zr4+-phosphate chemistry, and 3) the in situ grafting of electroactive polymers from the phosphorylated sites through the electrochemically mediated atom transfer radical polymerization (eATRP) at a negative potential, in the presence of the surface-attached alkyl halide as the initiator and the electroactive tag-conjugated acrylate as the monomer, respectively. Due to the electrochemically mediated polymerization, a large number of electroactive tags can be linked to each phosphorylated site, thereby greatly improving the detection sensitivity. This platform has been successfully applied to detect the activity of cAMP-dependent protein kinase (PKA) with a detection limit down to 1.63 mU mL-1. Results also demonstrate that it is highly selective and can be used for the screening of protein kinase inhibitors. The potential application of our platform for protein kinase activity detection in complex biological samples has been further verified using normal human serum and HepG2 cell lysate. Moreover, our platform is operationally simple, highly efficient and cost-effective, thus holding great potential in protein kinase detection and inhibitor screening.

Highly sensitive detection of sequence-specific DNA with morpholino-functionalized magnetic microspheres

In this work, a highly effective biosensor supported on morpholino-functionalized magnetic microspheres was investigated for the sequence-specific detection of DNA. Briefly, morpholino was first modified on the surface of magnetic microspheres through amido bonds, and then hybridized with target DNA in the ensuing step. Biotin-labeled signal probe DNA was designed to hybridize with the DNA nearby the 5′ end of sequence, and the biotin was used to bind with streptavidin-alkaline phosphatase (SA-ALP). L-Ascorbic acid-2-phosphate (AAP) was hydrolyzed by ALP to generate L-ascorbic acid, which could reduce resazurin to resorufin, resulting in a turn-on fluorescence signal. UV-vis absorbance spectroscopy was applied to prove the conjugation of ALP with signal probe DNA. Under optimal conditions, this biosensor displayed a good linear relationship between the fluorescence intensity and logarithm of single-stranded DNA concentrations in the range of 0.1 pM to 0.1 nM with a low detection limit of 9.32 fM. Moreover, fully complementary versus single-base mismatched, three-base mismatched and non-complementary DNA could be effectively distinguished. In addition, this novel approach rendered satisfactory analytical results for the determination of DNA in serum, thus exhibiting practical significance. These results demonstrated that this assay method displays excellent sensitivity and specificity for DNA detection and has great potential for practical applications.

Electrochemically mediated in situ growth of electroactive polymers for highly sensitive detection of double-stranded DNA without sequence-preference

The ability to directly detect double-stranded DNA (dsDNA) without sequence-preference continues to be a major challenge. Herein, we report an electrochemical method for the direct, highly sensitive detection of dsDNA based on the strand replacement of dsDNA by peptide nucleic acid (PNA) and the in situ growth of electroactive polymers through the surface-initiated electrochemically mediated atom transfer radical polymerization (SI-eATRP). Thiolated PNA molecules are firstly self-assembled onto gold electrode surface for the specific recognition of target dsDNA (dsDNA-T), which in turn leads to the formation of a high density of PNA/DNA heteroduplexes on the electrode surface for the subsequent attachment of ATRP initiators via the phosphate-Zr4+-carboxylate chemistry. By applying a negative potential to the electrode, the air-stable CuII deactivators can be reduced into the CuI activators so as to trigger the surface-initiated polymerization for the in situ growth of electroactive polymers. Due to the strand replacement of dsDNA by PNA, dsDNA can be directly detected without sequence-preference. Besides, the growth of polymers enables the modification of numerous electroactive probes, thereby greatly improving the electrochemical signal. Under optimal conditions, a good linearity between the electrochemical signal and the logarithm of dsDNA-T concentration over the range from 1.0 fM to 1.0nM, with a detection limit of 0.47 fM, can be obtained. Results indicate that it is highly selective, and holds high anti-interference capability in the presence of human serum samples. Therefore, this method offers great promises in providing a universal and efficient solution for the direct detection of dsDNA.