The liver has a major role in metabolism, digestion, detoxification and the elimination of various substances from the body. Liver diseases represent a global threat to health. Liver diseases can be caused by a variety of toxins, pathogens and chemicals. The ultimate end of liver injury can be liver fibrosis. An important category of liver disease is hepatocellular carcinoma. The proper managements of liver diseases require both rapid diagnosis and effective therapeutic tools. Nanotechnology is a new promising field can be used in medicine. The following chapter will discuss the various application of nanotechnology in management of variety of hepatic disorders.
Viral Hepatitis Detection by Nanotechnology
Quantum dots (QD) are inorganic fluorophores offering significant advantages over organic fluorophores conventionally used to label nucleic acids or proteins for optical detection [34, 35, 36]. They have several advantages as mentioned previously in addition to their compatibility with analyses of whole blood [34]. The major interest for their diagnostic use relates to the possibility of multiplexing [35].
Progress towards the ability to control the size and to functionalize the surface of nanoparticles has allowed the production of optically and chemically defined probes for the detection of biomolecules [37, 38].
Gold nanoparticles are another example of nanoparticles used as biosensors. A number of nanoparticle-based assays have been developed for biomolecular detection, with DNA- or protein-functionalized gold nanoparticles used as the target-specific probes (34, 38, 39). It is also possible to detect hybridization on a chip of oligonucleotides labeled by gold nanoparticles with a simple scanner [40]
This “scanometric” detection is simple and selective and allows the discrimination of a point mutation on the strand of DNA.
Furthermore, coupling this detection to a silver amplification method led to a level of sensitivity 100 times greater compared to fluorescent systems using confocal microscopy. In fact the presence of solution of silver and hydroquinone, the silver ions are reduced to the metal form at the surface of the gold nanoparticles thus allowing a 100,000 fold increase in the scanned signal intensity (40). DNA chips using gold nanoparticle probes and silver amplification have been developed for the detection of hepatitis A [41] and hepatitis E 42] viruses.
More recently, gold nanoparticles have been functionalized with antigens and combined in a sensitive immunoassay[43]. Thus the use of gold nanoparticles for optical detection should allow the development of new, compact, rapid and cost-effective tests.
The bio-barcode test is an ultrasensitive system of amplification and detection of nucleic acids or proteins [44-46] It uses two types of particles to perform purification, amplification and detection steps [47].
The first particle is a magnetic microparticle with a target recognition probe. In the case of nucleic acids, this probe is an oligonucleotide complementary to a statistically unique region of a target. For proteins, the recognition probe is a monoclonal antibody. The second particle is a nanoparticle bearing a recognition element that is complementary to another region of the target molecule with the result being to sandwich the target between the two types of particles. This recognition element would either be an oligonucleotide in case of a nucleic acid target or a polyclonal antibody if the target was a protein. In addition, this second particle carries with it hundreds of oligonucleotides referred to as barcodes. The application of a magnetic field leads to separate the complexed target from the rest of the sample. The barcode tags are then released either chemically or by heating and identified by a sensitive system of detection (for example the scanometric method). Thus the DNA barcode acts as a reporter of the targeted molecule which corresponds to a signal amplification. It is therefore possible to directly detect the DNA with zeptomolar sensitivity (10−21 M or 10 copies of DNA in a sample of 30 μl) making it as sensitive as PCR but avoiding the need for enzymes [45]. For proteins, this test reaches atto molar sensitivity (10−18 M), corresponding to a sensitivity one million times higher than that of classic ELISA tests [46, 47].
This technique was applied to the multiplex detection of the DNA of four viruses (HBV, Ebola, HIV and Smallpox). This proof-of-concept demonstration is a promising step towards highly multiplex assays [48]. The concept of the bio-barcode assay is particularly original and represents a potential alternative to the conventional PCR and ELISA based technologies.
One of the objectives for developing miniaturized systems is the concept of a truly integrated laboratory-type platform on a chip. A high performance rapid test based on a signal amplification system has been developed by the team of H. Lee for the HBV surface antigen (HBsAg) detection in plasma or serum specimens [49]. The integration of sample preparation in rapid tests remains a key issue [50].
A review published in Nature highlighted the impact of integrated miniaturized systems on public health, particularly in developing countries [51].
Nanotechnologies and microfluidics enable different measurements to be taken in parallel and the integration of several analytical steps from sample preparation through to detection in one individual miniaturized system [52, 53].
In 2006, Aytur et al. developed a biosensor using paramagnetic particles for the detection of antibodies directed against the Dengue virus in biological samples. This bioassay platform showed a very good correlation with measurements made with ELISA [54]. The bio-barcode approach was integrated into a microfluidic chip, marking an important step towards automization [55]. Recently, Liu et al. proposed an electrochemical immunosensor exploiting quantum dots to amplify the signal[56]. Another microfluidic immunoassay using quantum dots was proposed for the multiplex analysis of viruses HBV, HCV and HIV allowing their detection with a 50 times greater sensitivity compared to commercial ELISA kits[57].
“Nanolabels” can provide the means for signal amplification in optical or electrochemical biosensors while overcoming limitations of traditional labels [58]. Different strategies are in development; however the complete integration of all the steps of analysis and in particular of sample preparation onto the miniaturized support with a view to performing quantitative analysis remains very difficult [59].
The development of multiplex and flexible tests allowing the simultaneous analysis of pathogens presenting a transfusional risk remains a real challenge. To date, there still exist no platforms in diagnostic exploiting miniaturized systems that are as effective and robust as those based on ELISAs or target amplification as PCR or TMA. Finally, diagnostic microarrays for pathogen detection have been more limited than expected. This first generation of optical microarrays suffers from a low sensitivity and their combination to target amplification for DNA analysis impairs the cost/benefit ratio. Moreover the use of fluorescence technologies limits the possibility of multiplexing and requires costly reagents and equipment for signal analysis. The second generation of miniaturized systems combining micro- and nano-technologies should allow the development of highly adapted platforms. New developments in miniaturized technologies show promises but present many challenges. The questions of sensitivity, specificity, throughput and flexibility of these systems need to be evaluated in biological samples in order to be adapted to molecular diagnostic. Nanotechnologies and microfluidics offer new tools to improve the sample preparation step that remains difficult to integrate in a miniaturized platform. The signal amplification approaches could challenge those of target amplification. Non optical biosensors as magnetic or electrochemical biosensors are undergoing rapid development in diagnostic to allow a direct detection in real time of molecular targets. The combinations are infinite considering the multiple innovative techniques available. Emerging micro/nanoarrays have a potential to face emerging infectious agents but time will be needed before they could be considered as diagnostic devices adapted to blood testing.
Nanotechnology and advances in medicine
Dr Maysaa El Sayed Zaki
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