Enzyme-free Method Research Articles

Accurate and sensitive fluorescence detection of DNA based on G-quadruplex hairpin DNA

A facile and cost-effective fluorescence biosensor is constructed for the accurate and sensitive determination of DNA. The G-quadruplex-forming sequence in hairpin DNA sequence (H1) is originally locked. When target DNA is present, the hairpin structure of H1 can be unfolded and part of H1 hybridized with the target DNA to form a double-stranded DNA and a G-quadruplex. The hairpin DNA sequence (H2) hybridizes with the unfolded H1 and displaces the target DNA. Finally, the displaced target DNA again hybridizes with the H1 and initiates cycle amplification. Thus, the numerous G-quadruplexs at the H1 ends are formed, resulting in the fluorescent enhancement after binding with N-methyl mesoporphyrin IX (NMM). Lower background signal improves the accuracy of assay. The enzyme-free method can detect down to 40pM and a linear range of 3 orders of magnitude. Moreover, this strategy has an ability to discriminate the target DNA from even single-base mismatched or other sequence. The proposed method has potential applications in DNA-based molecular diagnostics.

Detecting 5-methylcytosine using an enzyme-free DNA strand exchange reaction without pretreatment under physiological conditions.

We present here a novel and efficient method for 5mC detection using a DNA strand exchange reaction (SER) strategy. This enzyme-free method needs no pre-treatment of target DNAs and can be adapted to most of the target duplexes under physiological conditions. The high sequence selectivity of this method can distinguish 5mC from normal cytosine in an accurate manner.

DNA tetrahedron and star trigon nanostructures for target recycling detection of nucleic acid.

Human immunodeficiency virus (HIV) is a retrovirus which attacks the human body's immune system and further leads to acquired immunodeficiency syndrome (AIDS). Nucleic acid detection is of great importance in the medical diagnosis of such diseases. Herein, we develop a simple and enzyme-free electrochemical method for the target recycling detection of nuclei acid. DNA tetrahedron and star trigon nanostructures are designed and constructed on the electrode interface for target capture and signal enrichment. This strategy is convenient and sensitive, with a limit of detection as low as 1 fM, and can also successfully distinguish single-base mismatched DNA. Therefore, the proposed method has a promising potential application for HIV DNA detection.

Evaluation of Tissue Homogenization to Support the Generation of GMP-Compliant Mesenchymal Stromal Cells from the Umbilical Cord.

Recent studies have demonstrated that the umbilical cord (UC) is an excellent source of mesenchymal stromal cells (MSCs). However, current protocols for extracting and culturing UC-MSCs do not meet current good manufacturing practice (cGMP) standards, in part due to the use of xenogeneic reagents. To support the development of a cGMP-compliant method, we have examined an enzyme-free isolation method utilizing tissue homogenization (t-H) followed by culture in human platelet lysate (PL) supplemented media. The yield and viability of cells after t-H were comparable to those obtained after collagenase digestion (Col-D). Importantly, kinetic analysis of cultured cells showed logarithmic growth over 10 tested passages, although the rate of cell division was lower for t-H as compared to Col-D. This slower growth of t-H-derived cells was also reflected in their longer population doubling time. Interestingly, there was no difference in the expression of mesenchymal markers and trilineage differentiation potential of cells generated using either method. Finally, t-H-derived cells had greater clonogenic potential compared to Col-D/FBS but not Col-D/PL and were able to maintain CFU-F capacity through P7. This bench scale study demonstrates the possibility of generating therapeutic doses of good quality UC-MSCs within a reasonable length of time using t-H and PL.

Guanine nanowire based amplification strategy: Enzyme-free biosensing of nucleic acids and proteins

Sensitive and specific detection of nucleic acids and proteins plays a vital role in food, forensic screening, clinical and environmental monitoring. There remains a great challenge in the development of signal amplification method for biomolecules detection. Herein, we describe a novel signal amplification strategy based on the formation of guanine nanowire for quantitative detection of nucleic acids and proteins (thrombin) at room temperature. In the presence of analytes and magnesium ions, the guanine nanowire could be formed within 10min. Compared to the widely used single G-quadruplex biocatalytic label unit, the detection limits are improved by two orders of magnitude in our assay. The proposed enzyme-free method avoids fussy chemical label-ling process, complex programming task, and sophisticated equipment, which might provide an ideal candidate for the fabrication of selective and sensitive biosensing platform.

A novel colorimetric sandwich aptasensor based on an indirect competitive enzyme-free method for ultrasensitive detection of chloramphenicol

Analytical methods for detection and quantitation of chloramphenicol in blood serum and foodstuffs arse highly in demand. In this study, a colorimetric sandwich aptamer-based sensor (aptasensor) was fabricated for sensitive and selective detection of chloramphenicol, based on an indirect competitive enzyme-free assay using gold nanoparticles (AuNPs), biotin and streptavidin. The designed aptasensor acquires characteristics of AuNPs, including large surface area and unique optical properties, and strong interaction of biotin with streptavidin. In the absence of chloramphenicol, the sandwich structure of aptasensor forms, leading to the observation of sharp red color. In the presence of target, functionalized AuNPs could not bind to 96-well plates, resulting in a faint red color. The fabricated colorimetric aptasensor exhibited high selectivity toward chloramphenicol with a limit of detection as low as 451pM. Moreover, the developed colorimetric aptasensor was successfully used to detect chloramphenicol in milk and serum with LODs of 697 and 601pM, respectively.

Enzyme-free detection of sequence-specific microRNAs based on nanoparticle-assisted signal amplification strategy

Developing direct and convenient methods for microRNAs (miRNAs) analysis is of great significance in understanding biological functions of miRNAs, and early diagnosis of cancers. We have developed a rapid, enzyme-free method for miRNA detection based on nanoparticle-assisted signal amplification coupling fluorescent metal nanoclusters as signal output. The proposed method involves two processes: target miRNA-mediated nanoparticle capture, which consists of magnetic microparticle (MMP) probe and CuO nanoparticle (NP) probe, and nanoparticle-mediated amplification for signal generation, which consists of fluorescent DNA–Cu/Ag nanocluster (NC) and 3-mercaptopropionic acid (MPA). In the presence of target miRNA, MMP probe and NP probe sandwich-capture the target miRNA via their respective complementary sequence. The resultant sandwich complex (MMP probe–miRNA–CuO NP probe) is separated using a magnetic field and further dissolved by acidolysis to turn CuO NP into a great amount of copper (II) ions (Cu2+). Cu2+ could disrupt the interactions between thiol moiety of MPA and the fluorescent Cu/Ag NCs by preferentially reacting with MPA to form a disulfide compound as intermediate. By this way, the fluorescence emission of the DNA–Cu/Ag NCs in the presence of MPA increases upon the increasing concentration of Cu2+, which is directly proportional to the amount of target miRNA. The proposed method allows quantitative detection of a liver-specific miR-221-5p in the range of 5pM to 1000pM with a detection limit of ∼0.73pM, and shows a good ability to discriminate single-base difference. Moreover, the detection assay can be applied to detect miRNA in cancerous cell lysates in excellent agreement with that from a commercial miRNA detection kit.

Novel Signal-Enhancing Approaches for Optical Detection of Nucleic Acids—Going beyond Target Amplification

Detection of low-abundance nucleic acids is a challenging task, which over the last two decades has been solved using enzymatic target amplification. Enzymatic synthesis enhances the signal so that diverse, scientifically and clinically relevant molecules can be identified and studied, including cancer DNA, viral nucleic acids, and regulatory RNAs. However, using enzymes increases the detection time and cost, not to mention the high risk of mistakes with amplification and data alignment. These limitations have stimulated a growing interest in enzyme-free methods within researchers and industry. In this review we discuss recent advances in signal-enhancing approaches aimed at nucleic acid diagnostics that do not require target amplification. Regardless of enzyme usage, signal enhancement is crucial for the reliable detection of nucleic acids at low concentrations. We pay special attention to novel nanomaterials, fluorescence microscopy, and technical advances in detectors for optical assessment. We summarize sensitivity parameters of the currently available assays and devices which makes this review relevant to the broad spectrum of researchers working in fields from biophysics, to engineering, to synthetic biology and bioorganic chemistry.

Target-driven self-assembly of stacking deoxyribonucleic acids for highly sensitive assay of proteins

In this paper, we report a new signal amplification strategy for highly sensitive and enzyme-free method to assay proteins based on the target-driven self-assembly of stacking deoxyribonucleic acids (DNA) on an electrode surface. In the sensing procedure, binding of target protein with the aptamer probe is used as a starting point for a scheduled cycle of DNA hairpin assembly, which consists of hybridization, displacement and target regeneration. Following numbers of the assembly repeats, a great deal of DNA duplexes can accordingly be formed on the electrode surface, and then switch on a succeeding propagation of self-assembled DNA concatemers that provide further signal enhancement. In this way, each target binding event can bring out two cascaded DNA self-assembly processes, namely, stacking DNA self-assembly, and therefore can be converted into remarkably intensified electrochemical signals by associating with silver nanoparticle-based readout. Consequently, highly sensitive detection of target proteins can be achieved. Using interferon-gamma as a model, the assay method displays a linear range from 1 to 500pM with a detection limit of 0.57pM, which is comparable or even superior to other reported amplified assays. Moreover, the proposed method eliminates the involvement of any enzymes, thereby enhancing the feasibility in clinical diagnosis.

Molecular Beacon-Based Fluorescent Assay for Specific Detection of Oversulfated Chondroitin Sulfate Contaminants in Heparin without Enzyme Treatment.

Oversulfated chondroitin sulfate (OSCS) is a harmful contaminant in the pharmaceutical heparin. The development of a rapid, convenient, sensitive, and selective method is required for routine analysis of OSCS in pharmaceutical heparin. Here we report a simple, rapid, sensitive, and enzyme-free method for detecting OSCS in heparin based on the competitive binding between OSCS and the adenosine-repeated molecular beacon (MB) stem to coralyne in the presence of Ca(2+) ions. The MB (A8-MB-A8) contains a 22-mer loop, a stem of a pair of 8-mer adenosine (A) bases, a fluorophore unit at the 5'-end, and a quencher at the 3'-end. The presence of coralyne promotes these A-A mismatches to form a hairpin-shaped MB. However, this kind of MB is incapable of differentiating between heparin and OSCS because they both exhibit strong electrostatic attraction with coralyne. This study found that while Ca(2+) ions can efficiently suppress the negative charges of heparin, they do not neutralize the negative charge of OSCS. Thus, in the presence of Ca(2+) ions, OSCS can remove coralyne from the MB stem, initiating fluorescence of the MB. Under optimal conditions (10 nM A8-MB-A8, 800 nM coralyne, and 0.5 mM Ca(2+) ions), the proposed system can detect 0.01% w/w OSCS in heparin in under 5 min without enzyme treatment. This study also validates the practicality of the proposed system to determine 0.01% w/w OSCS in the pharmaceutical heparin.

An enzyme-free electrochemical method for the determination of E. coli using Fe3O4 nanocomposites with a SiO2 shell modified by ferrocene

An electrochemical immunoassay method is developed for determining the strain of Escherichia coli ATCC 25922 with the use of electroactive nanocomposite particles based on magnetite Fe3O4 coated by SiO2 modified with ferrocene as a signal label. A sensitive, direct, and easily measured analytical response is obtained by cyclic voltammetry. The developed method enables the determination of bacterial cells at a minimum concentration of 2.3 × 103 CFU/mL. The analytical range is 2.3 × 103–2.3 × 107 CFU/mL; the detection limit is 2.5 × 102 CFU/mL. The required sample volume is 0.5 mL, and the analysis time is 40 min.

Enzyme-free amplified detection of microRNA using target-catalyzed hairpin assembly and magnesium ion-dependent deoxyribozyme

As microRNAs (miRNAs) are aberrantly expressed in a variety of cancers, detecting them precisely is of great importance. Here we constructed a sensitive and selective enzyme-free sensing platform for miRNA detection based on target-catalyzed hairpin assembly and magnesium ion-dependent deoxyribozyme (Mg2+-dependent DNAzyme). This sensing method introduces two amplification circuits simultaneously and shows a low detection limit of 1 pmol/L. This enzyme-free method is especially preferred because of its facility and economy. Furthermore, this amplified sensor shows high selectivity for discriminating perfectly complementary target and other mismatched RNAs. Therefore, the established strategy could be used as a simple, sensitive and selective method for target miRNA detection.

Hybridization chain reaction modulated DNA-hosted silver nanoclusters for fluorescent identification of single nucleotide polymorphisms in the let-7 miRNA family

A simple microRNA (miRNA) detection system based on hybridization chain reaction (HCR) has been developed using highly fluorescent DNA-hosted silver (Ag) nanoclusters. In this assay, a new type of hairpin DNA probe (MB1) containing a poly-cytosine nucleotide loop is designed and used as one of the HCR monomers, which is also demonstrated to be an ideal template for in situ synthesis of highly fluorescent Ag nanoclusters. Correspondingly, another HCR monomer (MB2) contains a poly-guanine nucleotide sticky end. Two monomers are stable to coexist in solution until the introduction of the initiator strand (let-7a) triggers a cascade of hybridization events that yields nicked double helices analogous. By taking advantage of HCR, a small amount of let-7a leads to the conformational change of a large amount of MB1, which results in the decrease of fluorescent signal greatly. Overall, this label-free, enzyme-free method allows the sensitive detection of let-7a with high specificity towards single nucleotide polymorphisms in the let-7 miRNA family. In addition, the simple “mix and measure” assay can be extended to detect other types of targets upon slight modification, and thus provides a tool for the early diagnosis and risk assessment of malignancy.

097 Non-enzymatic cryo-isolation of viable Islets from juvenile porcine pancreas after storage for >6 months at −135 °C

Cryo-isolation of therapeutic cells such as pancreatic islets has been proposed as a novel new alternative to conventional enzymatic digestion of the pancreas (Taylor, Baicu., Transpl Proc. 43 (2011) 3181–3183. And Cell Transpl. 2013 (In Press)). Apart from avoiding the expense, inconsistencies and toxicity associated with the collagenase-digestion process, cryo-isolation embodies several inherent advantages including the convenience of biobanking for storage and shipping. This study investigates the feasibility of obtaining viable islets from juvenile porcine pancreas after storage at −135 °C for >6 months using the cryo-isolation technique. Pancreases were procured from juvenile pigs using approved procedures and processed using the cryo-isolation protocol. This involved: (1) Infiltrating islets in situ preferentially with 2M dimethyl sulfoxide as the cryoprotectant (CPA) via antegrade perfusion of the major arteries; (2) retrograde ductal infusion of water, and then (3) the entire pancreas was frozen solid to −160 °C and stored in the vapor phase of liquid nitrogen. After storage at −135 °C for 1 day to 8 months, pancreases were further processed as follows: The frozen pancreas was mechanically crushed and pulverized into small fragments which were then thawed, filtered and washed with RPMI 1640 culture medium to remove the CPA. Finally, the filtered effluent (cryo-isolate) was stained with dithizone for identification of intact islets, and with Syto13/Propidium iodide (PI) for fluorescence viability testing and assessed for function using a conventional glucose-stimulated insulin secretion (GSIS) assay. The cryo-isolate from pancreata stored for >6 months contained pancreatic fragments comprising discrete dithizone-positive islets embedded in a shell of dead amorphous material. The cryo-isolate product was similar to that obtained from frozen pancreases stored for 1 day in that there was an abundance of largely intact and viable (>90%) islets. Moreover, the cryo-isolated islets were typically larger (range 50–500 μm) than their counterparts isolated from juvenile pigs using conventional collagenase digestion techniques and demonstrated GSIS indices (>3), which was not significantly different from controls (fresh islets from collagenase digestion). An enzyme-free method of islet isolation relying on in situ cryopreservation of islets with simultaneous freeze-destruction of acinar tissue is feasible and proposed as a new and novel method that avoids the problems associated with conventional collagenase digestion methods and permits long-term biobanking of pancreases as a ready source of islets. Source of funding: This work was funded in part by a grant from NIH/NIDDK#1R43DK096773. Conflict of interest: The authors are, or have been, employed by Cell and Tissue Systems. mtaylor@celltissuesystems.com

Isolation and purification of human blood plasma proteins able to form potassium channels in artificial bilayer lipid membrane

Protein fraction able to induce K(+)-selective transport across bilayer lipid membrane was isolated from human blood plasma with the use of the detergent and proteolytic enzyme-free method developed at our laboratory. After addition of the studied sample to the artificial membrane in the presence of 100 mM KCl, a discrete current change was observed. No channel activity was recorded in the presence of calcium and sodium ions. Channel forming activity of fraction was observed only in the presence of K+. Using a threefold gradient of KCl in the presence of studied proteins the potassium-selective potential balanced by voltage of -29 mV was registered. This value is very close to the theoretical Nernst potential in this case. This means that the examined ion channel is cation-selective. According to data obtained with MS-MALDI-TOF/TOF and database NCBI three protein components were identified in isolated researched sample.

Phosphorothioate-based DNA recombination: an enzyme-free method for the combinatorial assembly of multiple DNA fragments.

Rational guided generation of protein chimeras has developed into an attractive approach in protein engineering for tailoring catalytic and biophysical properties of enzymes. Combinatorial recombination of structural elements or whole protein domains is still technically challenging due to sequence dependent biases diminishing the overall quality of resulting chimeric libraries. Since methods for generating such libraries are often limited by a low frequency of crossover points and suffer from challenges in handling, we developed the phosphorothioate-based DNA recombination method (PTRec). PTRec is an enzyme-free method and only requires a short stretch of four amino acids that is identical among the proteins to be recombined in order to define a single crossover point. In a PTRec-generated chimeric library that shuffled five domains of phytase using genes from three different species, 88% of 42 randomly picked and sequenced genes were efficiently recombined. Furthermore, PTRec is a technically simple, fast, and reliable method that can be used for domain- and exon-shuffling or recombination of DNA fragments in general.

Cryo-isolation: A Novel Method for Enzyme-Free Isolation of Pancreatic Islets Involving In Situ Cryopreservation of Islets and Selective Destruction of Acinar Tissue

Background A critical component of treating type I diabetes by transplantation is the availability of sufficient high-quality islets. Currently, islets can be obtained only by reliance on an expensive, inconsistent, and toxic enzyme digestion process. As an alternative, we hypothesize that cryobiologic techniques can be used for differential freeze destruction of the pancreas to release islets that are selectively cryopreserved in situ. Methods Pancreases were procured from juvenile pigs with the use of approved procedures. The concept of cryo-isolation is based on differential processing of the pancreas in 5 stages: 1) infiltrating islets in situ preferentially with a cryoprotectant (CPA) cocktail via antegrade perfusion of the major arteries; 2) retrograde ductal infusion of water (or saline solution) to fully distend the gland; 3) freezing the entire pancreas to −160°C, and stored in liquid nitrogen; 4) mechanically crushing and pulverizing the frozen pancreas into small fragments; and 5) thawing, filtering and washing the frozen fragments with RPMI 1640 culture medium to remove the CPA. Finally, the filtered effluent (cryo-isolate) was stained with dithizone for identification of intact islets, and samples were taken for static glucose- stimulated insullin release assessment. Results As predicted the cryo-isolated contained small fragments of residual tissue comprising an amorphous mass of acinar tissue with largely intact embedded islets. The degree of cleavage of the cryoprotected islets from the freeze-destroyed exocrine cells, was variable. Islets were typically larger than their counterparts isolated from juvenile pigs with conventional enzyme-digestion techniques. Functionally, the islets from replicate cryo-isolates responded to a glucose challenge with a mean stimulation index = 3.3 ± 0.7 (n = 3). Conclusions An enzyme-free method of islet isolation relying on in situ cryopreservation of islets with simultaneous freeze-destruction of acinar tissue is feasible and proposed as a novel method that avoids the problems associated with conventional collagenase digestion methods.

Enzyme-Free Method for Cultured Skin Grafting

Cultured skin autografting has promise to improve the prognosis of severe burn injury. Established methods for cultured epithelial cell autografting need enzymatic proteolytic reaction that have potential risks of losing cell surface structures and causing allergic reactions in the recipients. Therefore, we have developed an enzyme-free method of cultured skin autografting. Donor skin was obtained in 0.2- 0.3 mm thickness during an operation of debriding burned eschar. The skin was cut into pieces and spread on removable soft culture surface membrane in culture plates.Modified MCDB 153 medium with 10 mug/L epidermal growth factor and 150 mg/L bovine pituitary extract was used. The tissues were incubated at 37 C in a water-saturated atmosphere of 5% CO2 in air. After 3 weeks, the cultured tissue on the membrane was removed mechanically from the bottom of the plates and implanted on granulated recipientsites in upside down manner. The cultured auto-skin grafting was successfully performed in two patients with severe burn. The recipient sites were partially epithelized in 2 weeks and maintained thereafter. Our method is performed easily using partial thickness donor skin without enzymatic processes.