Universal liposomes: preparation and usage for the detection of mRNA

Dye-encapsulating liposomes can serve as signaling reagents in biosensors and biochemical assays in place of enzymes or fluorophores. Detailed here is the use and preparation of streptavidin-coupled liposomes which offer a universal approach to biotinylated target detection. The universal approach provides two advantages, i.e. only one type of liposome is necessary despite varying target and probe sequences and the hybridization event can take place in the absence of potential steric hindrance occurring from liposomes directly conjugated to probes. One objective of this work was to optimize the one-step conjugation of SRB-encapsulating liposomes to streptavidin using EDC. Liposome, EDC, streptavidin concentrations, and reaction times were varied. The optimal coupling conditions were found to be an EDC:carboxylated lipid:streptavidin molar ratio of 600:120:1 and a reaction time of 15 min. The second goal was to utilize these liposomes in sandwich hybridization microtiter plate-based assays using biotinylated reported probes as biorecognition elements. The assay was optimized in terms of probe spacer length, probe concentration, liposome concentration, and streptavidin coverage. Subsequently, the optimized protocol was applied to the detection of DNA and RNA sequences. A detection limit of 1.7 pmol L⁻¹ and an assay range spanning four orders of magnitude (5 pmol L⁻¹−50 nmol L⁻¹) with a coefficient of variation ≤5.8% was found for synthetic DNA. For synthetic RNA the LOQ was half that of synthetic DNA. A comparison was made to alkaline phosphatase-conjugated streptavidin for detection which yielded a limit of quantitation approximately 80 times higher than that for liposomes in the same system. Thus, liposomes and the optimized sandwich hybridization method are well suited for detecting single-stranded nucleic acid sequences and compares favorably to other sandwich hybridization schemes recently described in the literature. The assay was then used successfully for the clear detection of mRNA amplified by nucleic acid sequence-based amplification (NASBA) isolated from as little as one Cryptosporidium parvum oocyst. The detection of mRNA from oocysts isolated from various water sample types using immunomagnetic separation was also assessed. Finally, to prove the wider applicability and sensitivity of this universal method, RNA amplified from the atxA gene of Bacillus anthracis was detected when the input to the preceding NASBA reaction was as low as 1.2 pg. This highly sensitive liposome-based microtiter plate assay is therefore a platform technology allowing for high throughput and wide availability for routine clinical and environmental laboratory applications.     

Optimization of DNA-tagged liposomes for use in microtiter plate analyses

Dye-encapsulating unilamellar DNA oligonucleotide-tagged liposomes were prepared and characterized for use as signal-enhancing reagents in a microtiter plate sandwich-hybridization analyses of single-stranded RNA or DNA sequences. The liposomes were synthesized using the reversed-phase evaporation method and tagged with DNA oligonucleotides by adding cholesteryl-modified DNA reporter probes to the initial lipid mixture. Liposomes were prepared using probe coverages of 0.0013–0.103 mol% of the total lipid input, several hydrophobic and poly(ethylene glycol)-based spacers between the cholesteryl anchor and the probe, and liposome diameters ranging from 200 nm to 335 nm. Their signal enhancement functionality was compared by using them in microtiter plate sandwich-hybridization assays for the detection of single-stranded DNA sequences. In these assays, an optimal reporter probe concentration of 0.103 mol%, a liposome diameter of 274 nm, and a phospholipid concentration of 0.3 mM were found. The length between the cholesteryl anchor and the probe was optimal when a spacer composed of TEG+(CH₂O)₃ was used. Under optimal conditions, a detection limit of 0.5 nM for a truncated synthetic DNA sequence was found with a coefficient of variation of 4.4%. A 500-fold lower limit of detection using fluorescence was found using lysed dye-encapsulating liposomes versus a single fluorescein-labeled probe. Finally, when this method was applied to the detection of atxA RNA extracted from E.coli SG12036-pIu121 and amplified using NASBA, a minimum extracted concentration of RNA of 1.1×10⁻⁷ μg/μL was found.     

Multi-analyte single-membrane biosensor for the serotype-specific detection of Dengue virus

A multi-analyte biosensor based on nucleic acid hybridization and liposome signal amplification was developed for the rapid serotype-specific detection of Dengue virus. After RNA amplification, detection of Dengue virus specific serotypes can be accomplished using a single analysis within 25 min. The multi-analyte biosensor is based on single-analyte assays (see Baeumner et al (2002) Anal Chem 74:1442–1448) developed earlier in which four analyses were required for specific serotype identification of Dengue virus samples. The multi-analyte biosensor employs generic and serotype-specific DNA probes, which hybridize with Dengue RNA that is amplified by the isothermal nucleic acid sequence based amplification (NASBA) reaction. The generic probe (reporter probe) is coupled to dye-entrapping liposomes and can hybridize to all four Dengue serotypes, while the serotype-specific probes (capture probes) are immobilized through biotin–streptavidin interaction on the surface of a polyethersulfone membrane strip in separate locations. A mixture of amplified Dengue virus RNA sequences and liposomes is applied to the membrane and allowed to migrate up along the test strip. After the liposome-target sequence complexes hybridize to the specific probes immobilized in the capture zones of the membrane strip, the Dengue serotype present in the sample can be determined. The amount of liposomes immobilized in the various capture zones directly correlates to the amount of viral RNA in the sample and can be quantified by a portable reflectometer. The specific arrangement of the capture zones and the use of unlabeled oligonucleotides (cold probes) enabled us to dramatically reduce the cross-reactivity of Dengue virus serotypes. Therefore, a single biosensor can be used to detect the exact Dengue serotype present in the sample. In addition, the biosensor can simultaneously detect two serotypes and so it is useful for the identification of possible concurrent infections found in clinical samples. The various biosensor components have been optimized with respect to specificity and sensitivity, and the system has been ultimately tested using blind coded samples. The biosensor demonstrated 92% reliability in Dengue serotype determination. Following isothermal amplification of the target sequences, the biosensor had a detection limit of 50 RNA molecules for serotype 2, 500 RNA molecules for serotypes 3 and 4, and 50,000 molecules for serotype 1. The multi-analyte biosensor is portable, inexpensive, and very easy to use and represents an alternative to current detection methods coupled with nucleic acid amplification reactions such as electrochemiluminescence, or those based on more expensive and time consuming methods such as ELISA or tissue culture.     

Optimization of DNA-tagged dye-encapsulating liposomes for lateral-flow assays based on sandwich hybridization

A novel protocol for the synthesis of dye-encapsulating liposomes tagged with DNA oligonucleotides at their outer surface was developed. These liposomes were optimized for use as signal enhancement agents in lateral-flow sandwich-hybridization assays for the detection of single-stranded RNA and DNA sequences. Liposomes were synthesized using the reverse-phase evaporation method and tagged with oligonucleotides by adding cholesteryl-modified DNA probes to the initial lipid mixture. This resulted in a greatly simplified protocol that provided excellent control of the probe coverage on the liposomes and cut the preparation time from 16 hours to just 6 hours. Liposomes were prepared using probe concentrations ranging from 0.00077 to 0.152 mol% of the total lipid, several hydrophobic and polyethylene glycol-based spacers between the cholesteryl anchor and the probe, and liposome diameters ranging from 208 nm to 365 nm. The liposomes were characterized by dynamic light scattering, visible spectroscopy, and fluorescence spectroscopy. Their signal enhancement functionality was compared by using them in lateral-flow optical biosensors for the detection of single-stranded DNA sequences. In these assays, an optimal reporter probe concentration of 0.013 mol%, liposome diameter of 315 nm, and liposome optical density of 0.4–0.6 at 532 nm were found. The spacer length between the cholesteryl anchor and the probe showed no significant effect on the signals in the lateral-flow assays. The results presented here provide important data for the general use of liposomes as labels in analytical assays, with specific emphasis on nucleic acid detection via lateral flow assays.     

Enzyme-amplified electrochemical hybridization assay based on PNA, LNA and DNA probe-modified micro-magnetic beads

In the present study, we investigated the properties of PNA and LNA capture probes in the development of an electrochemical hybridization assay. Streptavidin-coated paramagnetic micro-beads were used as a solid phase to immobilize biotinylated DNA, PNA and LNA capture probes, respectively. The target sequence was then recognized via hybridization with the capture probe. After labeling the biotinylated hybrid with a streptavidin–enzyme conjugate, the electrochemical detection of the enzymatic product was performed onto the surface of a disposable electrode. The assay was applied to the analytical detection of biotinylated DNA as well as RNA sequences. Detection limits, calculated considering the slope of the linear portion of the calibration curve in the range 0–2 nM were found to be 152, 118 and 91 pM, coupled with a reproducibility of the analysis equal to 5, 9 and 6%, calculated as RSD%, for DNA, PNA and LNA probes respectively, using the DNA target. In the case of the RNA target, the detection limits were found to be 51, 60 and 78 pM for DNA, PNA and LNA probes respectively.     

A nanogold-quenched fluorescence duplex probe for homogeneous DNA detection based on strand displacement

A nanogold-quenched fluorescence duplex probe has been developed for lighting up homogenous hybridization assays. This novel probe is constructed from two strands of different lengths, and labeled by nanogold and a fluorophore at the long-strand 5′-end and the short-strand 3′-end, respectively. The two tags are in close contact, resulting in complete quenching of the probe fluorescence. If perfectly complemented to the nanogold-labeled strand, a long target oligonucleotide would displace the short fluorophore-labeled strand, and as a result, restore the fluorescence. By using nanogold in the probe, an extremely high quenching efficiency (99.1%) and removal of free fluorophore-labeled strand is achieved. The signal-to-noise ratio and the detection limit (50 pmol L⁻¹) of homogenous assays are therefore improved significantly, in comparison with similar probes using organic acceptors. Moreover, the probe has a great inhibition effect on hybridization to a mismatched oligonucleotide. This effect provides the assay with a high specificity, and particularly the assay has great potential in applications for discriminating variations in sequences. The assay sensitivity could be markedly enhanced by using fluorescent materials in the signal strand that are brighter and not quenched by nucleobases.     

Selective fluorescence response and magnetic separation probe for 2,4,6-trinitrotoluene based on iron oxide magnetic nanoparticles

Despite the rapid development of nanomaterials and nanotechnology, it is still desirable to develop novel nanoparticle-based techniques which are cost-effective, timesaving, and environment-friendly, and with ease of operation and procedural simplicity, for assay of target analytes. In the work discussed in this paper, the dye fluorescein isothiocyanate (FITC) was conjugated to 1,6-hexanediamine (HDA)-capped iron oxide magnetic nanoparticles (FITC–HDA Fe₃O₄ MNPs), and the product was characterized. HDA ligands on the surface of Fe₃O₄ MNPs can bind 2,4,6-trinitrotoluene (TNT) to form TNT anions by acid–base pairing interaction. Formation of TNT anions, and captured TNT substantially affect the emission of FITC on the surface of the Fe₃O₄ MNPs, resulting in quenching of the fluorescence at 519 nm. A novel FITC–HDA Fe₃O₄ MNPs-based probe featuring chemosensing and magnetic separation has therefore been constructed. i.e. FITC–HDA Fe₃O₄ MNPs had a highly selective fluorescence response and enabled magnetic separation of TNT from other nitroaromatic compounds by quenching of the emission of FITC and capture of TNT in aqueous solution. Very good linearity was observed for TNT concentrations in the range 0.05–1.5 μmol?L^?1, with a detection limit of 37.2 nmol?L^?1 and RSD of 4.7 % (n?=?7). Approximately 12 % of the total amount of TNT was captured. The proposed methods are well-suited to trace detection and capture of TNT in aqueous solution.

Capillary electrophoresis for capture and concentrating of target nucleic acids by affinity gels modified to contain single-stranded nucleic acid probes

Selective capture and pre-concentration of target nucleic acids from relatively complicated samples may provide a method to facilitate introduction to a microfluidic-based detection system to improve detection limits. An acrylamide polymer gel modified with Acrydite^™ that contained 20mer oligonucleotide probe was prepared and loaded into a capillary column. The results indicated that the amount of probe DNA that was captured into the acrylamide was about 40% of the starting monomer, and different quantities of probe could therefore be coupled into the gel. The gel was passivated by pre-treatment with non-complementary DNA oligonucleotide to block non-selective adsorption sites, and the gel was determined to be stable for multiple cycles of use. The probe could hybridize with target sequences that were introduced by electrokinetic injection from a sample solution. The target could be freed from the polymer gel by use of a combination of heating, chaotropic salt and voltage conditions. Target capture efficiency was up to 90% when using samples that did not saturate probe sites in the columns, and recovery of target from the gel could be as high as 95%.     

Highly sensitive spectrofluorimetric determination of trace amounts of lecithin

A new spectrofluorimetric method was developed for the determination of trace amounts of lecithin using the ciprofloxacin (CIP)–terbium (Tb³⁺) ion complex as a fluorescent probe. In a buffer solution at pH=5.60, lecithin can remarkably reduce the fluorescence intensity of the CIP–Tb³⁺ complex at λ=545 nm. The reduced fluorescence intensity of the Tb³⁺ ion is proportional to the concentration of lecithin. Optimum conditions for the determination of lecithin were also investigated. The linear range and detection limit for the determination of lecithin were 1.0×10⁻⁶–3.0×10⁻⁵ mol L⁻¹ and 3.44×10⁻⁷ mol L⁻¹, respectively. This method is simple, practical, and relatively free of interference from coexisting substances. Furthermore, it has been successfully applied to assess lecithin in serum samples.      

Detection of oncoprotein platelet-derived growth factor using a fluorescent signaling complex of an aptamer and TOTO

There have recently been advances in the application of aptamers, a new class of nucleic acids that bind specifically with target proteins, as protein recognition probes for biomedical study. The development of a signaling aptamer with the capability of simple and rapid real-time detection of disease-related proteins has attracted increasing interest. We have recently reported a new protein-detection strategy using a signaling aptamer based on a DNA molecular light-switching complex, [Ru(phen)₂(dppz)]²⁺. In this work we have used the commercially available DNA-intercalating dye, TOTO, to replace [Ru(phen)₂(dppz)]²⁺ for detection of oncoprotein platelet-derived growth factor BB (PDGF-BB), a potential cancer marker. Taking advantage of the high affinity of the aptamer to PDGF-BB and the sensitive fluorescence change of the aptamer–TOTO signaling complex on protein binding, PDGF-BB was detected in physiological buffer with high selectivity and sensitivity. The detection limit was 0.1 nmol L⁻¹, which was better than that of other reported aptamer-based methods for PDGF-BB, including that using [Ru(phen)₂(dppz)]²⁺. The method is very simple with no need for covalent labeling of the aptamer or probe synthesis. It facilitates wide application of the signaling mechanism to the analysis and study of cancer markers and other proteins.      

Colorimetric quantification of mRNA expression in rare tumour cells amplified by multiple ligation-dependent probe amplification

An enzyme-linked oligonucleotide assay (ELONA) for quantification of mRNA expression of five genes involved in breast cancer, extracted from isolated rare tumour cells and amplified by multiplex ligation-dependent probe amplification (MLPA) is presented. In MLPA, a multiplex oligonucleotide ligation assay is combined with a PCR reaction in which all ligation products are amplified by use of a single primer pair. Biotinylated probes complementary to each of the target sequences were immobilised on the surface of a streptavidin-coated microtitre plate and exposed to single-stranded MLPA products. A universal reporting probe sequence modified with horseradish peroxidase (URP–HRP) and complementary to a universal primer used during the MLPA step was further added to the surface-bound duplex as a reporter probe. Simultaneous addition of anchoring probe and target, followed by addition of reporter probe, rather than sequential addition, was achieved with no significant effect on sensitivity and limits of detection, but considerably reduced the required assay time. Detection limits as low as 20 pmol L⁻¹, with an overall assay time of 95 min could be achieved with negligible cross-reactivity between probes and non-specific targets present in the MLPA-PCR product. The same MLPA-PCR product was analysed using capillary electrophoresis, the technique typically used for analysis of MLPA products, and good correlation was observed. The assay presented is easy to carry out, relatively inexpensive, rapid, does not require sophisticated instrumentation, and enables quantitative analysis, making it very promising for the analysis of MLPA products.     

A novel microfluidic flow-injection analysis device with fluorescence detection for cation sensing. Application to potassium

A microfabricated device has been developed for fluorimetric detection of potassium ions without previous separation. It is based on use of a fluorescent molecular sensor, calix–bodipy, specially designed to be sensitive to and selective for the target ion. The device is essentially made of a Y-shape microchannel moulded in PDMS fixed on a glass substrate. A passive mixer is used for mixing the reactant and the analyte. The optical detection arrangement uses two optical fibres, one for excitation by a light-emitting diode, the other for collection of the fluorescence. This system enabled the flow-injection analysis of the concentration of potassium ions in aqueous solutions with a detection limit of 0.5 mmol L⁻¹ and without interference with sodium ions. A calibration plot was constructed using potassium standard solutions in the range 0–16 mmol L⁻¹, and was used for the determination of the potassium content of a pharmaceutical pill. Figure Photography of the microfluidic channel showing the ridges in the PDMS substrate at the top of the channel     

Metal-enhanced fluorescent probes based on silver nanoparticles and its application in IgE detection

In this paper, a novel metal plasmon coupled with an aptamer–nucleotide hybridized probe was fabricated and applied for protein detection. The specific aptamer and single-strand oligonucleotide were chemically bound to silver nanoparticles (AgNPs), and Cy5-labeled, complementary single-strand oligonucleotides were hybridized with the particle-bound oligonucleotides. The hybridized DNA duplexes were regarded as rigid rods that separated the fluorophore Cy5 and the surface of AgNPs to reduce the competitive quenching. Using a model system comprising human immunoglobulin E (IgE) as the analyte and goat antihuman IgE as immobilized capture antibody on glass slides, we demonstrate that the detection performance of the synthetic probe was superior to the aptamer-based fluorescent probes. The results showed a good linear correlation for human IgE in the range from 10 ng/ml to 6.25 μg/ml. The detection limit obtained was 1 ng/ml, which was 50 times lower than that using Cy5 oligonucleotide/aptamer hybrid duplex (Probe2) due to the metal-enhanced fluorescence effect. This new strategy opens the possibility for the preparation of high-sensitivity detection probes based on metal nanoparticles.     

A rapid biosensor for viable <Emphasis Type="Italic">B. anthracis</Emphasis> spores

A simple membrane-strip-based biosensor assay has been combined with a nucleic acid sequence-based amplification (NASBA) reaction for rapid (4 h) detection of a small number (ten) of viable B. anthracis spores. The biosensor is based on identification of a unique mRNA sequence from one of the anthrax toxin genes, the protective antigen (pag), encoded on the toxin plasmid, pXO1, and thus provides high specificity toward B. anthracis. Previously, the anthrax toxins activator (atxA) mRNA had been used in our laboratory for the development of a biosensor for the detection of a single B. anthracis spore within 12 h. Changing the target sequence to the pag mRNA provided the ability to shorten the overall assay time significantly. The vaccine strain of B. anthracis (Sterne strain) was used in all experiments. A 500-L sample containing as few as ten spores was mixed with 500 L growth medium and incubated for 30 min for spore germination and mRNA production. Thus, only spores that are viable were detected. Subsequently, RNA was extracted from lysed cells, selectively amplified using NASBA, and rapidly identified by the biosensor. While the biosensor assay requires only 15 min assay time, the overall process takes 4 h for detection of ten viable B. anthracis spores, and is shortened significantly if more spores are present. The biosensor is based on an oligonucleotide sandwich-hybridization assay format. It uses a membrane flow-through system with an immobilized DNA probe that hybridizes with the target sequence. Signal amplification is provided when the target sequence hybridizes to a second DNA probe that has been coupled to liposomes encapsulating the dye sulforhodamine B. The amount of liposomes captured in the detection zone can be read visually or quantified with a hand-held reflectometer. The biosensor can detect as little as 1 fmol target mRNA (1 nmol L^–1). Specificity analysis revealed no cross-reactivity with 11 organisms tested, among them closely related species such as B. cereus, B. megaterium, B. subtilis, B. thuringiensis, Lactococcus lactis, Lactobacillus plantarum, and Chlostridium butyricum. Also, no false positive signals were obtained from nonviable spores. We suggest that this inexpensive biosensor is a viable option for rapid, on-site analysis providing highly specific data on the presence of viable B. anthracis spores.     

Label-free detection of nucleic acids by turn-on and turn-off G-quadruplex-mediated fluorescence

In this study we have used two fluorescent probes, tetrakis(diisopropylguanidino)-zinc-phthalocyanine (Zn-DIGP) and N-methylmesoporphyrin IX (NMM), to monitor the reassembly of “split” G-quadruplex probes on hybridization with an arbitrary “target” DNA. According to this approach, each split probe is designed to contain half of a G-quadruplex-forming sequence fused to a variable sequence that is complementary to the target DNA. Upon mixing the individual components, both base-pairing interactions and G-quadruplex fragment reassembly result in a duplex–quadruplex three-way junction that can bind to fluorescent dyes in a G-quadruplex-specific way. The overall fluorescence intensities of the resulting complexes were dependent on the formation of proper base-pairing interactions in the duplex regions, and on the exact identity of the fluorescent probe. Compared with samples lacking any “target” DNA, the fluorescence intensities of Zn-DIGP-containing samples were lower, and the fluorescence intensities of NMM-containing samples were higher on addition of the target DNA. The resulting biosensors based on Zn-DIGP are therefore termed “turn-off” whereas the biosensors containing NMM are defined as “turn-on”. Both of these biosensors can detect target DNAs with a limit of detection in the nanomolar range, and can discriminate mismatched from perfectly matched target DNAs. In contrast with previous biosensors based on the peroxidase activity of heme-bound split G-quadruplex probes, the use of fluorescent dyes eliminates the need for unstable sensing components (H₂O₂, hemin, and ABTS). Our approach is direct, easy to conduct, and fully compatible with the detection of specific DNA sequences in biological fluids. Having two different types of probe was highly valuable in the context of applied studies, because Zn-DIGP was found to be compatible with samples containing both serum and urine whereas NMM was compatible with urine, but not with serum-containing samples.     

Dendrimer-based biosensor for chemiluminescent detection of DNA hybridization

We report on a highly sensitive chemiluminescent (CL) biosensor for the sequenc-specific detection of DNA using a novel bio barcode DNA probe modified with gold nanoparticles that were covered with a dendrimer. The modified probe is composed of gold nanoparticles, a dendrimer, the CL reagent, and the DNA. The capture probe DNA was immobilized on magnetic beads covered with gold. It first hybridizes with the target DNA and then with one terminal end of the signal DNA on the barcoded DNA probe. CL was generated by adding H₂O₂ and Co(II) ions as the catalyst. The immobilization of dendrimer onto the gold nanoparticles can significantly enhance sensitivity and gives a detection limit of 6 fmol L^-1 of target DNA.

Rapid fluorimetric assay for primary amine groups in water samples

Bond Elut C₁₈ solid-phase extraction cartridges were used for pre-concentration followed by derivatization with o-phthaldialdehyde-N-acetylcysteine (OPA-NAC) of primary amines in water. Optimal conditions were: conditioning the cartridges with borate buffer pH 10.4, retention of the primary amines, addition of the OPA-NAC(3.7 mmol L^–1) 1:1 molar ratio and borate buffer pH 8, elution of the isoindol with MeOH-borate buffer (9:1) pH 10.2 and fluorescence measurement. The equations of the calibration graphs for methylamine, ethylamine, propylamine, butylamine, pentylamine, and -phenylethylamine at _excitation=330 nm and _emission=440 nm, in the optimal conditions are presented. The solid-phase extraction procedure improved ten times the detection limits of the solution derivatization. Those values are in the 0.01–0.06 mg L^–1 interval in function of the amine. Also, it is possible to estimate the total primary aliphatic amine concentration in water, expressed as molar concentration of –NH₂ group or –NH₂-N mg L^–1. On the basis of these studies, the method was applied for the determination of primary amino groups in tap, ground, factory and source water samples.     

Electrochemical detection of miRNA-222 by use of a magnetic bead-based bioassay

MicroRNAs (miRNAs, miRs) are naturally occurring small RNAs (approximately 22 nucleotides in length) that have critical functions in a variety of biological processes, including tumorigenesis. They are an important target for detection technology for future medical diagnostics. In this paper we report an electrochemical method for miRNA detection based on paramagnetic beads and enzyme amplification. In particular, miR 222 was chosen as model sequence, because of its involvement in brain, lung, and liver cancers. The proposed bioassay is based on biotinylated DNA capture probes immobilized on streptavidin-coated paramagnetic beads. Total RNA was extracted from the cell sample, enriched for small RNA, biotinylated, and then hybridized with the capture probe on the beads. The beads were then incubated with streptavidin–alkaline phosphatase and exposed to the appropriate enzymatic substrate. The product of the enzymatic reaction was electrochemically monitored. The assay was finally tested with a compact microfluidic device which enables multiplexed analysis of eight different samples with a detection limit of 7 pmol L^?1 and RSD?=?15 %. RNA samples from non-small-cell lung cancer and glioblastoma cell lines were also analyzed.     

Toward hybridization assays without PCR using universal nanoamplicons

An innovative scheme for signal amplification using random tetramer-modified gold nanoparticles, termed “nanoamplicons,” has been developed for hybridization assay without PCR. Large numbers of nanoamplicons could be integrated onto one target, providing much greater amplification than the larger nanoparticles usually adopted. Using M13mp18 single-strand DNA as a target, this concept is shown to be a feasible approach to detecting 0.17 amol L⁻¹ DNA without target amplification, based on microgravimetric detection of the adsorption of the probe–target–nanoamplicons complex via thiol–gold binding. To our knowledge, this method has a sensitivity that is close to that of PCR and superior to those of nanoparticle-based methods reported previously. Additionally, this novel nanoamplicon could be prepared in the same way and used for all diagnostic tests; such universality would make the nanoamplicons highly advantageous for the generalization and standardization of bioassays, and when applying this new technology in clinical laboratories. Figure A novel signal amplification method for DNA detection with subattomolarsensitivity has been developed using random tetramer-modified gold nanoparticlesas nanoamplicons, which are easily prepared with high uniformity and can be universally adaptedto any sequences