Noncompetitive phage anti-immunocomplex real-time polymerase chain reaction for sensitive detection of small molecules

Immuno polymerase chain reaction (IPCR) is an analytical technology based on the excellent affinity and specificity of antibodies combined with the powerful signal amplification of polymerase chain reaction (PCR), providing superior sensitivity to classical immunoassays. Here we present a novel type of IPCR termed phage anti-immunocomplex assay real-time PCR (PHAIA-PCR) for the detection of small molecules. Our method utilizes a phage anti-immunocomplex assay (PHAIA) technology in which a short peptide loop displayed on the surface of the M13 bacteriophage binds specifically to the antibody-analyte complex, allowing the noncompetitive detection of small analytes. The phagemid DNA encoding this peptide can be amplified by PCR, and thus, this method eliminates hapten functionalization or bioconjugation of a DNA template while providing improved sensitivity. As a proof of concept, two PHAIA-PCRs were developed for the detection of 3-phenoxybenzoic acid, a major urinary metabolite of some pyrethroid insecticides, and molinate, a herbicide implicated in fish kills. Our results demonstrate that phage DNA can be a versatile material for IPCR development, enabling universal amplification when the common element of the phagemid is targeted or specific amplification when the real time PCR probe is designed to anneal the DNA encoding the peptide. The PHAIA-PCRs proved to be 10-fold more sensitive than conventional PHAIA and significantly faster using magnetic beads for rapid separation of reactants. The assay was validated with both agricultural drain water and human urine samples, showing its robustness for rapid monitoring of human exposure or environmental contamination.     

Highly adaptable and sensitive protease assay based on fluorescence resonance energy transfer

Proteases are widely used in analytical sciences and play a central role in several widespread diseases. Thus, there is an immense need for highly adaptable and sensitive assays for the detection and monitoring of various proteolytic enzymes. We established a simple protease fluorescence resonance energy transfer (pro-FRET) assay for the determination of protease activities, which could in principle be adapted for the detection of all proteases. As proof of principle, we demonstrated the potential of our method using trypsin and enteropeptidase in complex biological mixtures. Briefly, the assay is based on the cleavage of a FRET peptide substrate, which results in a dramatic increase of the donor fluorescence. The assay was highly sensitive and fast for both proteases. The detection limits for trypsin and enteropeptidase in Escherichia coli lysate were 100 and 10 amol, respectively. The improved sensitivity for enteropeptidase was due to the application of an enzyme cascade, which leads to signal amplification. The pro-FRET assay is highly specific as even high concentrations of other proteases did not result in significant background signals. In conclusion, this sensitive and simple assay can be performed in complex biological mixtures and can be easily adapted to act as a versatile tool for the sensitive detection of proteases.     

Carbon Nanotubes Multifunctionalized by Rolling Circle Amplification and Their Application for Highly Sensitive Detection of Cancer Markers

There are still challenges for the development of multifunctional carbon nanotubes (CNTs). Here, a multiwalled carbon nanotube (MWCNT)‐based rolling circle amplification system (CRCAS) is reported which allows in situ rolling circle replication of DNA primer on the surface of MWCNTs to create a long single‐strand DNA (ssDNA) where a large number of nanoparticles or proteins could be loaded, forming a nano‐biohybridized 3D structure with a powerful signal amplification ability. In this strategy, the binding ability of proteins, hybridization, replication ability of DNA, and the catalytical ability of enzymes are integrated on a single carbon nanotube. The CRCAS is then used to develop colorimetric and chemiluminescent assays for the highly sensitive and specific detection of cancer protein markers, alpha‐fetoprotein (AFP) and prostate specific antigen (PSA). The colorimetric CRCAS assay is 4000 times more sensitive than a conventional enzyme‐linked immunosorbent assay (ELISA), and its concentration range is 10 000 times wider. Control experiments show that as low as 10 pg mL^?1 AFP or PSA could be detected even in the presence of interfering protein markers with a more than 10⁵‐fold greater concentration in the sample, demonstrating the high specificity of the CRCAS assay. The limit of detection of the chemiluminescent CRCAS assays for AFP and PSA are 5 fg mL^?1 (70 aM) and 10 fg mL^?1 (0.29 fM), respectively, indicating that the sensitivity is much higher than that of the colorimetric CRCAS assay. Importantly, CRCAS works well with real biological samples.     

Functionalised gold nanoparticles for selective induction of in vitro apoptosis among human cancer cell lines

The interaction of citrate- and polyethylene imine (PEI)-functionalised gold nanoparticles (GNP) with cancer cell lines with respect to the cellular response was studied. It was found that GNP/citrate nanoparticles were able to induce apoptosis in human carcinoma lung cell lines A549, but GNP/PEI did not show any reduction in the viability of the cells in human breast cancer cell line MCF-7 and A549 cell lines. FACS data confirmed that the number of apoptotic cells increased with increase in the concentration of GNP/citrate nanoparticles. Decline in cellular expansion and changes in the nuclear morphology were noted after the treatment of GNP/citrate nanoparticles on A549 cell lines, which itself is a direct response for stress induction. The induction of cellular apoptosis was further confirmed by DNA fragmentation assay. These data confirm the potential of GNP/citrate nanoparticle to evoke cell-specific death response in the A549 cell lines.     

A Dual‐Responsive Mesoporous Silica Nanoparticle for Tumor‐Triggered Targeting Drug Delivery

A novel pH‐ and redox‐ dual‐responsive tumor‐triggered targeting mesoporous silica nanoparticle (TTTMSN) is designed as a drug carrier. The peptide RGDFFFFC is anchored on the surface of mesoporous silica nanoparticles via disulfide bonds, which are redox‐responsive, as a gatekeeper as well as a tumor‐targeting ligand. PEGylated technology is employed to protect the anchored peptide ligands. The peptide and monomethoxypolyethylene glycol (MPEG) with benzoic‐imine bond, which is pH‐sensitive, are then connected via “click” chemistry to obtain TTTMSN. In vitro cell research demonstrates that the targeting property of TTTMSN is switched off in normal tissues with neutral pH condition, and switched on in tumor tissues with acidic pH condition after removing the MPEG segment by hydrolysis of benzoic‐imine bond under acidic conditions. After deshielding of the MPEG segment, the drug‐loaded nanoparticles are easily taken up by tumor cells due to the exposed peptide targeting ligand, and subsequently the redox signal glutathione in tumor cells induces rapid drug release intracellularly after the cleavage of disulfide bond. This novel intelligent TTTMSN drug delivery system has great potential for cancer therapy.     

Supramolecular Recognition‐Mediated Layer‐by‐Layer Self‐Assembled Gold Nanoparticles for Customized Sensitivity in Paper‐Based Strip Nanobiosensors

Herein, a smart supramolecular self‐assembly‐mediated signal amplification strategy is developed on a paper‐based nanobiosensor to achieve the sensitive and customized detection of biomarkers. The host–guest recognition between β‐cyclodextrin‐coated gold nanoparticles (AuNPs) and 1‐adamantane acetic acid or tetrakis(4‐carboxyphenyl)porphyrin is designed and applied to the layer‐by‐layer self‐assembly of AuNPs at the test area of the strip. Thus, the amplified platform exhibits a high sensitivity with a detection limit at subattogram levels (approximately dozens of molecules per strip) and a wide dynamic range of concentration over seven orders of magnitude. The applicability and universality of this sensitive platform are demonstrated in clinically significant ranges to measure carcinoembryonic antigen and HIV‐1 capsid p24 antigen in spiked serum and clinical samples. The customized biomarker detection ability for the on‐demand needs of clinicians is further verified through cycle incubation‐mediated controllable self‐assembly. Collectively, the supramolecular self‐assembly amplification method is suitable as a universal point‐of‐care diagnostic tool and can be readily adapted as a platform technology for the sensitive assay of many different target analytes.     

DNA-functionalized monolithic hydrogels and gold nanoparticles for colorimetric DNA detection

Highly sensitive and selective DNA detection plays a central role in many fields of research, and various assay platforms have been developed. Compared to homogeneous DNA detection, surface-immobilized probes allow washing steps and signal amplification to give higher sensitivity. Previously research was focused on developing glass or gold-based surfaces for DNA immobilization; we herein report hydrogel-immobilized DNA. Specifically, acrydite-modified DNA was covalently functionalized to the polyacrylamide hydrogel during gel formation. There are several advantages of these DNA-functionalized monolithic hydrogels. First, they can be easily handled in a way similar to that in homogeneous assays. Second, they have a low optical background where, in combination with DNA-functionalized gold nanoparticles, even ～0.1 nM target DNA can be visually detected. By using the attached gold nanoparticles to catalyze the reduction of Ag+, as low as 1 pM target DNA can be detected. The gels can be regenerated by a simple thermal treatment, and the regenerated gels perform similarly to freshly prepared ones. The amount of gold nanoparticles adsorbed through DNA hybridization decreases with increasing gel percentage. Other parameters including the DNA concentration, DNA sequence, ionic strength of the solution, and temperature have also been systematically characterized in this study.     

Fluorescence near gold nanoparticles for DNA sensing

We investigated fluorescence quenching and enhancement near gold nanoparticles (GNP) of various sizes using fluorescently labeled hairpin DNA probes of different lengths. A closed hairpin caused intimate contact between the fluorophore and the gold, resulting in an efficient energy transfer (quenching). Upon hybridization with complementary DNA, the DNA probes were stretched yielding a strong increase in fluorescence signal. By carefully quantifying the amount of bound fluorescent probes and the GNP concentrations, we were able to determine the quenching and enhancement efficiencies. We also studied the size and distance dependence theoretically, using both FDTD simulations and the Gersten-Nitzan model and obtained a good agreement between experiments and theory. On the basis of experimental and theoretical studies, we report over 96.8% quenching efficiency for all particle sizes tested and a maximal signal increase of 1.23 after DNA hybridization. The described results also demonstrate the potential of gold nanoparticles for label free DNA sensing.     

Mixed Nanosized Polymeric Micelles as Promoter of Doxorubicin and miRNA‐34a Co‐Delivery Triggered by Dual Stimuli in Tumor Tissue

Dual stimuli‐sensitive mixed polymeric micelles (MM) are developed for co‐delivery of the endogenous tumor suppressor miRNA‐34a and the chemotherapeutic agent doxorubicin (Dox) into cancer cells. The novelty of the system resides in two stimuli‐sensitive prodrugs, a matrix metalloproteinase 2 (MMP2)‐sensitive Dox conjugate and a reducing agent (glutathione, GSH)‐sensitive miRNA‐34a conjugate, self‐assembled in a single particle decorated with a polyethylene glycol corona for longevity, and a cell‐penetrating peptide (TATp) for enhanced intracellular delivery. The MMP2‐sensitivity of the system results in threefold higher cytotoxicity in MMP2‐overexpressing HT1080 cells compared to low MMP2‐expressing MCF7 cells. Cellular internalization of Dox increases by more than 70% after inclusion of TATp to the formulation. MMP2‐sensitive MM also inhibits proliferation and migration of HT1080 cells. Moreover, GSH‐sensitive MM allows for an efficient downregulation of Bcl2, survivin, and notch1 (65%, 55%, and 46%, respectively) in HT1080 cells. Combination of both conjugates in dual sensitive MM reduces HT1080 cell viability to 40% and expression of Bcl2 and survivin. Finally, 50% cell death is observed in 3D models of tumor mass. The results confirm the potential of the MM to codeliver miRNA‐34a and doxorubicin triggered by dual stimuli inherent of tumor tissues.     

Detection of specific sequences in RNA using differential adsorption of single-stranded oligonucleotides on gold nanoparticles

We have used the disparity in adsorption rates for single- and double-stranded RNA on ionically coated gold nanoparticles suspended in a colloid to design a rapid sequence identification assay. Unlabeled target RNA and a probe sequence are mixed prior to exposure to the gold nanoparticles to enable efficient hybridization. We have designed assays based on either color changes or fluorescence that are sensitive to a few picomoles of target. Single-base mutations on RNA sequences can be detected even in complex oligonucleotide mixtures. The assay requires less than 10 min so that RNA degradation problems are avoided.     

Sensitive detection of bacterial DNA by magnetic nanoparticles

This work presents sensitive detection of bacterial genomic DNA using a magnetic nanoparticle-based substrate-free method. For the first time, such a method is employed for detection of a clinically relevant analyte by implementing a solid-phase-based molecular probing and amplification protocol that can be executed in 80 min. The molecular detection and amplification protocol is presented and verified on samples containing purified genomic DNA from Escherichia coli cells, showing that as few as 50 bacteria can be detected. This study moves the use of volume-amplified magnetic nanoparticles one step further toward rapid, sensitive, and selective infectious diagnostics.     

Surface plasmon resonance biosensor for rapid label-free detection of microribonucleic acid at subfemtomole level

Microribonucleic acids (miRNAs) have been linked with various regulatory functions and disorders, such as cancers and heart diseases. They, therefore, present an important target for detection technologies for future medical diagnostics. We report here a novel method for rapid and sensitive miRNA detection and quantitation using surface plasmon resonance (SPR) sensor technology and a DNA*RNA antibody-based assay. The approach takes advantage of a novel high-performance portable SPR sensor instrument for spectroscopy of surface plasmons based on a special diffraction grating called a surface plasmon coupler and disperser (SPRCD). The surface of the grating is functionalized with thiolated DNA oligonucleotides which specifically capture miRNA from a liquid sample without amplification. Subsequently, an antibody that recognizes DNA*RNA hybrids is introduced to bind to the DNA*RNA complex and enhance sensor response to the captured miRNA. This approach allows detection of miRNA in less than 30 min at concentrations down to 2 pM with an absolute amount at high attomoles. The methodology is evaluated for analysis of miRNA from mouse liver tissues and is found to yield results which agree well with those provided by the quantitative polymerase chain reaction (qPCR).     

Lighting Up MicroRNA in Living Cells by the Disassembly of Lock‐Like DNA‐Programmed UCNPs‐AuNPs through the Target Cycling Amplification Strategy

Intracellular microRNAs imaging based on upconversion nanoprobes has great potential in cancer diagnostics and treatments. However, the relatively low detection sensitivity limits their application. Herein, a lock‐like DNA (LLD) generated by a hairpin DNA (H1) hybridizing with a bolt DNA (bDNA) sequence is designed, which is used to program upconversion nanoparticles (UCNPs, NaYF₄@NaYF₄:Yb, Er@NaYF₄) and gold nanoparticles (AuNPs). The upconversion emission is quenched through luminescence resonance energy transfer (LRET). The multiple LLD can be repeatedly opened by one copy of target microRNA under the aid of fuel hairpin DNA strands (H2) to trigger disassembly of AuNPs from the UCNP, resulting in the lighting up of UCNPs with a high detection signal gain. This strategy is verified using microRNA‐21 as model. The expression level of microRNA‐21 in various cells lines can be sensitively measured in vitro, meanwhile cancer cells and normal cells can be easily and accurately distinguished by intracellular microRNA‐21 imaging via the nanoprobes. The detection limit is about 1000 times lower than that of the previously reported upconversion nanoprobes without signal amplification. This is the first time a nonenzymatic signal amplification method has been combined with UCNPs for imaging intracellular microRNAs, which has great potential for cancer diagnosis.     

Transducing Protease Activity into DNA Output for Developing Smart Bionanosensors

DNA can process information through sequence‐based reorganization but cannot typically receive input information from most biological processes and translate that into DNA compatible language. Coupling DNA to a substrate responsive to biological events can address this limitation. A two‐component sensor incorporating a chimeric peptide‐DNA substrate is evaluated here as a protease‐to‐DNA signal convertor which transduces protease activity through DNA gates that discriminate between different input proteases. Acceptor dye‐labeled peptide‐DNAs are assembled onto semiconductor quantum dot (QD) donors as the input gate. Addition of trypsin or chymotrypsin cleaves their cognate peptide sequence altering the efficiency of Förster resonance energy transfer (FRET) with the QD and frees a DNA output which interacts with a tetrahedral output gate. Downstream output gate rearrangement results in FRET sensitization of a new acceptor dye. Following characterization of component assembly and optimization of individual steps, sensor ability to discriminate between the two proteases is confirmed along with effects from joint interactions where potential for cross‐talk is highest. Processing multiple bits of information for a sensing outcome provides more confidence than relying on a single change especially for the discrimination between different targets. Coupling other substrates to DNA that respond similarly could help target other types of enzymes.     

DNA-capped mesoporous silica nanoparticles as an ion-responsive release system to determine the presence of mercury in aqueous solutions

We have developed DNA-functionalized silica nanoparticles for the rapid, sensitive, and selective detection of mercuric ion (Hg(2+)) in aqueous solution. Two DNA strands were designed to cap the pore of dye-trapped silica nanoparticles. In the presence of ppb level Hg(2+), the two DNA strands are dehybridized to uncap the pore, releasing the dye cargo with detectable enhancements of fluorescence signal. This method enables rapid (less than 20 min) and sensitive (limit of detection, LOD, 4 ppb) detection, and it was also able to discriminate Hg(2+) from twelve other environmentally relevant metal ions. The superior properties of the as-designed DNA-functionalized silica nanoparticles can be attributed to the large loading capacity and highly ordered pore structure of mesoporous silica nanoparticles, as well as the selective binding of thymine-rich DNA with Hg(2+) . Our design serves as a new prototype for metal-ion sensing systems, and it also has promising potential for detection of various targets in stimulus-release systems.     

Luminescent lanthanide nanoparticles as labels in DNA microarrays for quantification of methyl tertiary butyl ether degrading bacteria

We report application of lanthanide nanoparticles for DNA quantification in a microarray platform as a substitute for conventional organic fluorophores. A non-PCR based DNA microarray assay for quantifying bacteria capable of biodegrading methyl tertiary-butyl ether (MTBE) was demonstrated. Probe DNA was immobilized on a glass surface, hybridized with biotinylated target DNA and subsequently incubated with Neutravidin-biofunctionalized nanoparticles. The fluorescence spot intensities, measured by a commercial laser scanner, show a linear relationship (R2 = 0.98) with bacterial 16S rDNA over a range of target DNA concentrations, while the background fluorescence remained low. In addition, nanoparticles fluorescence shows a stronger intensity than Quasar570 (Cy3). Present sensitivity of the assay is 10 pM of target DNA. The selectivity of the DNA-nanoparticle-probes to discriminate a non-target DNA with two base pairs mismatch in the 16S rDNA gene sequence was shown. The use of Eu:Gd2O3 nanoparticles as biolabels provides a relatively non-toxic, inexpensive, rapid and sensitive alternative to the materials currently used in DNA microarrays.     

Visualizing Human Telomerase Activity with Primer‐Modified Au Nanoparticles

Telomerase is over‐expressed in over 85% of all known human tumors. This renders the enzyme a valuable biomarker for cancer diagnosis and an important therapeutic target. The most widely used telomeric repeat amplification protocol (TRAP) assay has been questioned for telomerase detection. It is reported that human telomerase activity can be visualized by using primer‐modified Au nanoparticles. The working principle is based on the elongated primers conjugated to the gold nanoparticle (AuNP) surface, which can fold into a G‐quadruplex to protect the AuNPs from the aggregation. The developed simple and sensitive colorimetric assay can measure telomerase activity down to 1 HeLa cell µL^?1. More importantly, this assay can be easily extended to high‐throughput and automatic format. The AuNP‐TS method is PCR‐free and therefore avoids the amplification‐related errors and becomes more reliable to evaluate telomerase activity. This assay has also been used for initial screening of telomerase inhibitors as anticancer drug agents.     

Plasmonic coupling interference (PCI) nanoprobes for nucleic acid detection

A label-free approach using plasmonic coupling interference (PCI) nanoprobes for nucleic acid detection using surface-enhanced Raman scattering (SERS) is described. To induce a strong plasmonic coupling effect, a nanonetwork of silver nanoparticles with the Raman label located between adjacent nanoparticles is assembled by Raman-labeled DNA-locked nucleic acid (LNA) duplexes. The PCI method then utilizes specific nucleic acid sequences of interest as competitor elements for the Raman-labeled DNA strands to interfere the formation of nanonetworks in a competitive binding process. As a result, the plasmonic coupling effect induced through the formation of the nanonetworks is significantly diminished, resulting in a reduced SERS signal. The potential of the PCI technique for biomedical applications is illustrated by detecting single-nucleotide polymorphism (SNP) and microRNA sequences involved in breast cancers. The results of this study could lead to the development of nucleic acid diagnostic tools for biomedical diagnostics and biosensing applications using SERS detection.