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Kewal K. Jain- Applications of Nanobiotechnology in Clinical Diagnostics

Kewal K. Jain- Applications of Nanobiotechnology in Clinical Diagnostics

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Applications of Nanobiotechnology inClinical Diagnostics
Kewal K. Jain
Background:
Nanobiotechnologies are being applied tomolecular diagnostics and several technologies are indevelopment.
 Methods:
This review describes nanobiotechnologiesthat are already incorporated in molecular diagnostics orhave potential applications in clinical diagnosis. Se-lected promising technologies from published literatureas well as some technologies that are in commercialdevelopment but have not been reported are included.
 Results:
Nanotechnologies enable diagnosis at the sin-gle-cell and molecule levels, and some can be incorpo-rated in current molecular diagnostic methods, such asbiochips. Nanoparticles, such as gold nanoparticles andquantum dots, are the most widely used, but variousother nanotechnological devices for manipulation at thenanoscale as well as nanobiosensors are also promisingfor potential clinical applications.
Conclusions:
Nanotechnologies will extend the limitsof current molecular diagnostics and enable point-of-care diagnostics, integration of diagnostics with thera-peutics, and development of personalized medicine.Although the potential diagnostic applications are un-limited, the most important current applications areforeseen in the areas of biomarker discovery, cancerdiagnosis, and detection of infectious microorganisms.Safety studies are needed for in vivo use. Because of itsclose interrelationships with other technologies, nano-biotechnology in clinical diagnosis will play an impor-tant role in the development of nanomedicine in thefuture.
© 2007 American Association for Clinical Chemistry
Nanomolecular diagnostics, the use of nanobiotechnologyin molecular diagnostics
(1)
, and nanobiotechnology, theuse of various nanotechnologies and their applications inlife sciences
(2)
offer new options for clinical diagnosticprocedures. No recognized classification system for nano-diagnostics is currently in use, but a proposed systemwith the main categories of technologies is shown in Table1. Further descriptions of some of these categories areprovided in subsequent sections of this review. Nanopar-ticle biolabels are listed in Table 2 and are described indetail elsewhere
(3)
. Molecular diagnostics is an essentialpart of the development of personalized medicine, whichfeatures point-of-care performance of diagnostic proce-dures. This report focuses on the application of thesetechnologies in the clinical laboratory setting. Interrela-tionships of nanotechnology and molecular diagnosticsand their role in nanomedicine as well as personalizedmedicine are shown schematically in Fig. 1.Nanoscale probes are suitable for detailed analysis ofreceptors, pores, and other components of living cells thatare on a nanoscale. Thus nanotechnology can be used toimprove PCR as well as provide non-PCR methods forrapid diagnostics. Advantages of applying nanotechnol-ogy to molecular diagnostics are that only small amountsof sample material are needed and that diagnostic teststhat use nanoscale particles as tags or labels are faster andmore sensitive
(4)
. Nanoparticles can also be used tocombine diagnostics with therapeutics.
Nanotechnology-Based Biochips and Microarrays
Nanotechnology on a chip is a new paradigm for totalchemical analysis systems
(5)
. The ability to make chem-ical and biological information easier and less costly toobtain will impact molecular diagnostics and healthcare.Some examples of devices that incorporate nanotechnol-ogy-based biochips and microarrays are nanofluidic ar-rays and protein nanobiochips. These devices can beadapted for point-of-care use.One of the more promising uses of nanofluidic devicesis isolation and analysis of individual biomolecules, suchas DNA. This capability could lead to new detectionschemes for cancer. One such device entails the construc-tion of silicon nanowires on a substrate, or chip, usingstandard photolithographic and etching techniques, fol-lowed by a chemical oxidation step that converts the
 Jain PharmaBiotech, Basel, Switzerland.Address correspondence to the author at: Jain PharmaBiotech, Blaesiring7, CH-4057 Basel, Switzerland. Fax 4161-692-44-61; e-mail jain@pharmabiotech.ch.Received April 24, 2007; accepted August 28, 2007.Previously published online at DOI: 10.1373/clinchem.2007.090795
Clinical Chemistry
53:112002–2009 (2007)
Oak Ridge Conference
2002
 
nanowires into hollow nanotubes
(6)
. With this process,the investigators can reliably create nanotubes with diam-eters as small as 10 nm, although devices used for biomolecule isolation contain nanotubes with diametersof 50 nm. Trapping DNA molecules requires a deviceconsisting of a silicon nanotube connecting 2 parallelmicrofluidic channels. Electrodes provide a source ofcurrent used to drive DNA into the nanotubes. Each timea single DNA molecule moves into the nanotube, theelectrical current suddenly changes. The current returnsto its baseline value when the DNA molecule exits thenanotube. Nanofluidic technology is expected to have broad applications in systems biology, personalized med-icine, pathogen detection, drug development, and clinicalresearch.Protein microarrays for the study of protein functionare not widely used, in part because of the challenges inproducing proteins to spot on the arrays. Protein microar-rays can be generated by printing complementary DNAsonto glass slides and then translating target proteins withmammalian reticulocyte lysate
(7)
. Epitope tags fused tothe proteins allow them to be immobilized in situ. Thisprocedure obviates the need to purify proteins, avoidsprotein stability problems during storage, and capturessufficient protein for functional studies. This technologyhas been used to map pairwise interactions among 29human DNA replication initiation proteins and to reca-pitulate the regulation of Cdt1 binding to select replica-tion proteins and map the geminin-binding domain.
Nanotechnology-Based Cytogenetics
Cytogenetics has been used mainly to describe the chro-mosome structure and identify abnormalities related todisease. The localization of specific gene probes by fluo-rescent in situ hybridization (FISH)
2
combined with con-ventional fluorescence microscopy has reached its limit.Molecular cytogenetics in now enhanced by use of nano- biotechnology, e.g., atomic force microscopy and quan-tum dot (QD) FISH.Both atomic force microscopy and scanning near-fieldoptical microscopy have been used to obtain local infor-mation from G-bands and chromosomal probes. The finalresolution allows a more precise localization comparedwith standard techniques, and the extraction of very smallamounts of chromosomal DNA by the scanning probe ispossible. This method is also focused on the combinationof biochemical and nanomanipulation techniques, whichenable both nanodissection and nanoextraction of chro-mosomal DNA.The photostability and narrow emission spectra ofnonorganic QD fluorophores make them desirable candi-dates for the use of FISH to study the expression ofspecific mRNA transcripts. A method for direct QDlabeling of modified oligonucleotide probes using strepta-vidin and biotin interactions increased sensitivity ofmultiple-label FISH
(8)
. This technique also gives excel-lent histological results for FISH combined withimmunohistochemistry.QD’s broad absorption spectra allowed different col-ored probes specific for distinct subnuclear genetic se-quences to be simultaneously excited with a single exci-tation wavelength and imaged free of chromaticaberrations in a single exposure. A rapid method for thedirect multicolor imaging of multiple subnuclear geneticsequences uses novel QD-based FISH. A Texas red dyegamma-satellite probe produces fluorescent foci at theperiphery of interphase nucleus and labels every centro-mere in metaphase chromosomes
(9)
.
 Application of Nanoparticles for Tracking Stem Cells
A superparamagnetic iron oxide (SPIO) nanoparticle isemerging as an ideal probe for noninvasive cell tracking.However, its low intracellular labeling efficiency haslimited its use and stimulated interest in the developmentof new labeling strategies.The use of 200-nm perfluorocarbon nanoparticles tolabel endothelial progenitor cells taken from human um- bilical cord blood enables in vivo progenitor cell detection by MRI
(10)
. The MRI scanner can be tuned to the specificfrequency of the fluorine compound in the nanoparticles,and only the nanoparticle-containing cells are visible in
2
Nonstandard abbreviations: FISH, fluorescent in situ hybridization; QD,quantum dot; SPIO, superparamagnetic iron oxide; RSV respiratory syncytialvirus; GMA, glycidyl methacrylate; SPR, surface plasmon resonance; SERS,surface enhanced Raman scattering.
Table 1. Classification of categories of nanodiagnostic technologies.
Nanoscale visualization, e.g., atomic force microscopy, scanningprobe microscopyNanoparticle biolabelsNanotechnology-based biochips/microarraysNanoparticle-based nucleic acid diagnosticsNanoproteomic-based diagnosticsBiobarcode assaysNanopore technologyDNA nanomachines for molecular diagnosticsNanoparticle-based immunoassaysNanobiosensorsCombinations of multiple diagnostic technologies
Table 2. Nanoparticle biolabels.
Quantum dots as labelsSilver nanoparticle labelsSilica nanoparticles for labeling antibodiesSERS nanotagsDNA nanotagsFluorescent lanthanide nanorodsPerfluorocarbon nanoparticlesOrganic nanoparticles as biolabels
Clinical Chemistry
53, No. 11, 2007
2003
 
the scan. This method eliminates any background signals,which often interfere with medical imaging. Moreover,the lack of interference enables measurement of very lowamounts of the labeled cells and estimation of theirnumber on the basis of the brightness of the image.Because several perfluorocarbon compounds are avail-able, different types of cells could be labeled with differ-ent compounds, injected, and then detected separately bytuning the MRI scanner to the individual frequency ofeach cell type. This technology offers significant advan-tages over other cell-labeling technologies in develop-ment. Laboratory tests showed that the cells retained theirusual surface markers and that they were still functionalafter the labeling process. The labeled cells were shown tomigrate to and incorporate into blood vessels formingaround tumors in mice. These methods could soon enableresearchers and physicians to use unique signatures fromthe ingested nanoparticle beacons to directly track cellsused in medical treatments. Such tracking ability couldprove useful for monitoring tumors and diagnosing aswell as treating cardiovascular problems.
Nanoscale Single-Cell or Molecule Identification
Nanotechnology has facilitated the development of meth-ods for detection of single cells or a few molecules.Nanolaser scanning confocal spectroscopy, with the capa- bility of single-cell resolution, can be used to identifypreviously unknown properties of certain cancer cells thatdistinguishes them from closely related nonpathogeniccells
(11)
. Nanoproteomics, the application of nanobio-technology to proteomics, can enable detection of a singlemolecule of protein
(2)
. Biobarcode assays enable detec-tion in body fluids of miniscule amounts of proteins thatcannot be detected by conventional methods
(12)
. A2-dimensional method for mass spectrometry in solutionis based on the interaction between a nanometer-scalepore and analytes
(13)
. An applied electric current is usedto force charged molecules (such as single-stranded DNA)one at a time into the nanopore, which is only 1.5 nm at itssmallest point. As the molecules pass through the chan-nel, the current flow is reduced in proportion to the size ofeach individual chain, allowing its mass to be easilyderived. This single-molecule analysis technique couldprove useful for the real-time characterization of biomarkers.
 Application of Nanoparticles for Discovery of Biomarkers
Currently available molecular diagnostic technologieshave been used to detect biomarkers of various diseases.Nanotechnology has refined the detection of biomarkers.Some biomarkers also form the basis of innovative mo-lecular diagnostic tests. The physicochemical characteris-tics and high surface areas of nanoparticles make themideal candidates for developing biomarker-harvestingplatforms. Given the variety of nanoparticle technologiesthat are available, it is feasible to tailor nanoparticlesurfaces to selectively bind a subset of biomarkers andsequester them for later study using high-sensitivity pro-teomic tests
(14)
. Biomarker harvesting is an underuti-lized application of nanoparticle technology and is likelyto undergo substantial growth. Functional polymer-coated nanoparticles can be used for quick detection of biomarkers and DNA separation.
Nanoparticles for Molecular Diagnostics
Several nanoparticles have been used for diagnostics. Ofthese, the most frequently used are gold nanoparticles,QDs, and magnetic nanoparticles.
Fig. 1. This scheme shows the interre-lationship of various technologies thatcontribute to clinical nanodiagnostics.
These technologies also contribute to de-velopment of nanomedicine under the con-cept of personalized medicine.
2004
Jain: Nanobiotechnology in Clinical Diagnostics

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