using nanoparticles in hcv diagnosis
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Introduction• Nanoparticles are structures in the nanometer size range, which
can present different shapes (such as spheres, cylinders, and tubes) , compositions, charges, surface modifications.
• Nanoparticles can be made of materials of diverse chemical nature, the most common being metals, metal oxides, silicates, polymers, carbon, lipids, and biomolecules.
zirconia (ZrO2) nanoparticles (100nm) are inorganic oxides with thermal stability and chemical inertness without toxicity.
Silica (SiO2) nanoparticles (40-50nm) are robust inorganic materials, which have been used to prepare sandwich-type immunosensors.
Magnetic nanoparticles which can be manipulated using magnetic field. Such particles commonly consist of magnetic elements such as iron, nickeland cobalt and their chemical compounds
Gold nanoparticles (AuNPs) are spheres with a typical diameter of approximately 2–50 nm. They exhibit a unique phenomenon known as Surface Plasmon Resonance (SPR), which refers to the collective oscillation of the free electrons on the surface of AuNPs, when they are hit by light.
SPR is responsible for their intense red color. Colloidal solution of AuNPs (20 nm diameter) would appear an intense red color at absorbance 520 nm
Quantum dots (QDs), which are semiconductor nanocrystals of semiconductor materials with Cadmium selenide /zinc sulphide core-shell. QDs are able to improve the performance of fluoroassays by offering a number of benefits over traditional organic fluorophores (eg. acridine orange), such as high brightness, longer fluorescence lifetime, better photostability, as well as narrow and symmetric emission spectrum.
When AuNPs come close together, e.g., due to electrostatic attraction by another moiety, or due to alteration of medium ionic strength, Plasmon-Plasmon coupling occurs and change in solution color from red to blue .
The signal generated in AuNPs-based molecular diagnostic assays can be detected by various strategies including:
colorimetric, scanometric, light-scattering, electrochemical and electrical, nanometal surface energy transfer (NSET), surface enhanced Raman scattering (SERS) and laser diffraction strategies .
AuNPs colorimetric methods are quite attractive in diagnostic applications and, usually employ:
Positively charged (cationic) unmodified AuNPs.
Negatively charged particles, and can be further categorized into:
unmodified (no probes) AuNPs non-cross linking (one probe) AuNPs ,cross-linking (two probes) AuNPs ,
Direct detection of unamplified HCV RNA using unmodified gold nanoparticles
• AuNPs-based colorimetric method has been used to directly detect unamplified HCV RNA extracted from clinical specimens .
• Colorimetric assay using unmodified citrate -coated AuNPs (negatively charged) based on the fact that single stranded DNA (ssDNA) adsorbs on citrate-coated AuNPs,
• This adsorption increases the negative charge on the AuNPs leading to increased repulsion between the particles, thus preventing aggregation.
• The adsorption of ssDNA on AuNPs occurs due to the fact that ssDNA can uncoil and expose its nitrogenous bases. The attractive electrostatic forces between the bases and the AuNPs allow adsorption of the ssDNA.
On the other hand, double-stranded DNA (dsDNA) does not adsorb on AuNPs due to the repulsion between its negatively-charged phosphate backbone and the negatively-charged coating of
citrate ions on the surfaces of the AuNPs.
Therefore, when AuNPs are added to a saline solution containing the target ( HCV RNA) and its complementary unlabeled primer) ssDNA), AuNPs aggregate (since the primers are not free to stabilize the AuNPs) and the solution color changes to blue.
However, in the absence of the target (HCV RNA) or the presence of a non-complementary target, the primers) ssDNA) are free to stabilize the AuNPs thus preventing their aggregation and the solution color remains red.
The steps of the assay:PCR tube contains the sample (the extracted RNA ) and hybrdization buffer (primer and NaCl).
The sample is denatured at 95 °C for 30 s and annealed at 59 °C for 30 s
15 nm AuNPs is added after cooling the mixture at room temperature for 10 min.
The photographs were taken after 1 min from the addition of the AuNPs.
Only one primer (unlabeled ss DNA probe) is used of 27 nucleotides long that targeting HCV RNA 5 UTR to all HCV genotypes and subtypes′
(a)HCV RNA negative samples (red) (b) HCV RNA positive
samples (blue)
The color of AuNPs solution is affected by four main factors which should be adequately optimized for best results:
•The concentrations of NaCl, • The concentration of 15-nm AuNP• The concentration of primer• The assay temperature.
•The addition of AuNPs directly after removal of the tubes from the thermal cycler resulted in false positive results.
•Increasing the time of the denaturation and annealing steps also increases the percentage of false positive results.
•The assay has a sensitivity of 92%, a specificity of 89% compared to real time RT PCR, and a detection limit of 50 copies/reaction.
Direct detection of unamplified HCV RNA using unmodified gold and modified
magnetic nanoparticles
In this study, using a two metallic nanoparticles based colorimetric platform (solution phase) which employs:
• Magnetic nanoparticles modified with HCV RNA-specific probe for capturing of HCV RNA in serum.
• unmodified positively charged (cationic) AuNPs for the direct detection of unamplified HCV RNA extracted from serum specimens.
• The cationic AuNPs bind to the negatively charged phosphate backbone of nucleic acids extracted from serum.
• The cationic AuNPs align closely along these molecules and thus color changed from red to blue.
In the absence of the target, the positively-charged AuNPs repel each other and the solution color remains red.
The steps of assay:• The extracted HCV RNA is captured with magnetic nanoparticles
conjugated to oligonucleotide specific to HCV RNA. The captured RNA is then washed and eluted for purification with the help of permanent magnet.
• addition of AuNPs and phosphate buffer to the purified RNA and the color of the tube is observed within 5 min.
• In the presence of HCV RNA target, alignment of the positively charged AuNPs onto the negatively charged phosphate backbone of HCV RNA occurred leading to nanoparticles aggregation and solution color changed from red to blue.
• However, in case of negative samples, no nucleic acid was present for alignment of the AuNPs and thus nanoparticles remained separated from each other due to repulsion between their positive charges and the solution color remained red.
The presence of phosphate buffer in the assay increased the aggregation capability of the AuNPs (for only the
positive samples)
The aggregation occurred by first alignment of AuNPs on phosphate backbone of one nucleic acid, which then binds
to phosphate ions and the latter binds to other AuNPs which then binds to other nucleic acid phosphate
backbone.
Therefore, several layers (1stRNA/AuNPs/Phosphate ions/AuNPs/2ndRNA) were formed in the positive samples
leading to AuNPs aggregation.
SEM of AuNPs in positive HCV sample. AuNPs aggregate on RNA
molecules in the presence of phosphate buffer.
SEM of AuNPs inthe absence of HCV RNA (no aggregation of
AuNPs).
The intensity of blue color is proportional to the HCV viral load.
The assay has high sensitivity and specificity of > 95%, and a detection limit of 15 IU/mL.
Detection of HCV antibodies using modified gold nanoparticles in Lateral flow rapid tests
This assay uses gold nanoparticles modified with a mixture of NS3, NS4 and core HCV antigens for detection HCV antibodies in clinical specimens.
• The a sample is applied to the sample well and the addition of the provided sample diluent immediately.
• HCV antigen- Gold conjugate embedded in the sample pad reacts with the HCV antibody present in serum or plasma, forming conjugate/HCV antibody complex.
• As the mixture is allowed to migrate along the test strip, the conjugate/HCV antibody complex is captured by an antibody-binding protein A immobilized on a membrane forming a colored band in the test region.
• A negative sample does not produce a test line due to the absence of Gold conjugate/HCV antibody complex.
• A colored control band in the control region appears at the end of the test procedure regardless of the test result.
• This control band is the result of Gold conjugate binding to an anti-HCV antibody immobilized on the membrane.
• The control line indicates that the Gold conjugate is functional• The absence of the control band indicates that the test is invalid
Detection of HCV antigen using nanoparticle-based DNA amplification in BCA protein chip
Mirkin’s group used a bio-barcode amplification (BCA) technique to amplify the signal in the protein detection approach.
• In the BCA method, the biochip detects barcode DNA rather than the target antigen.
• The present study performs ultrasensitive detection of protein (HCV antigen) by integrating an electrical approach utilizing MNPs and bio-barcode DNA to improve electrical current through nanogap electrodes from extremely low concentrations of target DNA.
To detect target HCV antigen in the sample by the barcode DNA amplification approach, two nanoparticles (gold nanoparticles and magnetic nanoparticles) and two set of biomolecules are prepared:
• One set contained two proteins (HCV monoclonal antibody (2B2), and HCV polyclonal antibodies (GP)), where the antigen can specifically bind to both antibodies.
• The second set contained three single-strand DNAs (capture DNA (CDNA), barcode DNA, and probe DNA (PDNA)).
• The barcode DNA is complementary to both CDNA and PDNA.• The sequences of CDNA, barcode DNA, and PDNA are 3-HSA10-
CCT AAT AAC-5, 5-GGA TTA TTG TTA AAT ATT GAT AAG GAT-3 and 3-TTA TAA CTA TTC CTA-A10-SH5, respectively.
With HCV antigen in the sample solution, AuNPs with polyclonal antibodies and barcode ssDNA are bound with MNPs with monoclonal antibodies through a sandwich binding between polyclonal antibodies, target HCV antigen, and monoclonal antibody.• Since MNPs have superparamagnetic properties, the AuNP–MNP conjugates can be held by a permanent magnet and cannot be washed away.• If the sample solution contains no target HCV antigen, the MNPs and AuNPs binding does not occur. • Consequently, the 0.3M PBS buffer solution washes away all the unbound AuNPs with barcode DNA.• Then washing with a 0.1M PBS buffer dehybridizes a large ratio of barcode DNA for target antigen amplification from AuNP–DNA and MNP–protein conjugate
• Using the magnetic separator, the barcode DNA is released from the hybridized AuNPs–DNA and MNPs–protein aqueous solution.
• The self-assembled monolayer of AuNPs was established on the silicon dioxide surface using APTMS.
• One end of the APTMS compound was to silanize the group substrate surface while the amine group at the other end was used to bind the AuNPs .
• Self-assembled multilayer AuNPs established through the hybridization of PDNA, CDNA, barcode DNA and AuNP conjugate
If DNA hybridization is not fully complementary, the top layer of AuNPs is washed away .
A electrical detection of protein using self-assembled multilayer AuNP onto SiO2/Si substrate between gold electrodes.
• The particle density of AuNPs over the nanogap surface increases as the concentration of HCV antigen increases.
• Although a target HCV antigen is intended for detection in the sample solution, barcode DNA is used instead of HCV antigen to establish multilayer gold nanoparticles for electrical detection in I–V curve measurements.
• The detection limit of the protein biochip is 1 pg/uL of target HCV antigen.
Label-free sandwich type of immunosensor for HCVcore antigen based on the use of gold nanoparticleson a nanostructured metal oxide surface
This study reports on the construction of a label-free electrochemical immunosensor for detecting the core antigen of the hepatitis C virus (HCV core antigen).
A glassy carbon electrode (GCE) (3 mm) is modified with a nanocomposite made from gold nanoparticles, zirconia nanoparticles and chitosan, and prepared by in situ reduction method.
AuNPs are synthesized on the surfaces of ZrO2 nanoparticles by chitosan in electrochemical immunosensor.
In parallel, a nanocomposite was synthesized from AuNPs, silica nanoparticles and chitosan, and conjugated to a secondary
antibody.
• AuNPs/ZrO2-Chits nanocomposites are dropped on the pretreated electrode GCE and then Ab1 solution (primary anti core HCV) is add to immerse The AuNPs/ ZrO2-Chits-modified electrode
• Next, the resulting electrode is washed with phosphate buffer solution to remove the physically absorbed Ab1,
• finally bovine serum albumin ( BSA) is added to the obtained nanocomposites block possible remaining active sites on the AuNPs and avoid the nonspecific Adsorption.
For detection, the immunosensor is incubated with ( sample solution ) and then washed.
Subsequently, the electrode is incubated with Ab2/AuNPs/SiO2-Chits bioconjugate solution and then washed to remove nonspecifically bound conjugates.
Detection HCV core antigen depend on the current response of the immunosensor where the current response increases when the concentration of HCV core antigen increases . An sandwich type of immunosensor displays high sensitivity to the HCV core antigen with a detection limit of 0.17 ng mL−1 .
Quantum-dots-based detection of hepatitis C virus (HCV) NS3 using RNA
aptamer on chip
This study is the first report on HCV capturing and imaging using a QDs-based target-oriented specific RNA aptamer strategy. Appling
the HCV-specific RNA aptamer of QDs-based nanoparticle on chip, thereby developing a novel HCV diagnostic method.
With the aid of nanoparticle quantum dots (QDs) with carboxyl group as an imaging probe, and 5-end-amine-modified RNA oligonucleotide as a capturing probe, target HCV NS3 was visually detected on chip.
• Aptamers are single-stranded nucleic acids that detect high affinity binding to a variety of targets, small molecules, peptide, glycan, protein and biological molecules.
The specific oligonucleotide, termed RNA aptamer, which can bind NS3 protein with high affinity, has been elucidated. (H2N-5-GCAGUAGUGUAUAGGC-3)
• The conjugation of QDs and aptamer was performed by reaction between free carboxyl groups at the surface of QDs605 with the amino group of NS3 aptamer.
• The Subcloned, expressed and purified HCV NS3 protein is prepared.• HCV protein detection on chip: (i) immobilization of HCV protein on The linker terminated glass chip (ii) binding of QDs-aptamer conjugates on an immobilized chip; (iii) washing for discard of unspecific binding; and (iv) The chip is analyzed by a confocal laser scanning microscope, the
QDs-NS3 treated conjugates showed high fluorescent signals on the chip. No fluorescence signal was detected with BSA as the reference protein, due to its lack of affinity with the QDs-NS3 aptamer.
The detection level of viral protein NS3 by conventional method such as ELISA was approximately 450 μgmL−1. The QDs-based RNA aptamer sensor system showed the lower detection limit (5 ng mL−1) for evaluation of HCV viral protein NS3.
