dale m. willard- nanoparticles in bioanalytics

8/3/2019 Dale M. Willard- Nanoparticles in bioanalytics

http://slidepdf.com/reader/full/dale-m-willard-nanoparticles-in-bioanalytics 1/4

Inorganic compounds with nanometer dimensions displayproperties unlike the bulk material due to electronic con-finement effects. The result is a new class of compoundscalled nanoparticles. Since the 1970s, primarily physicistsand material scientists investigated nanoparticles to iden-tify and exploit their unique properties. Applications orig-inally centred on electronic and optical devices, however,research is now proving that some of the most exciting ap-plications will be in bioanalytics. This brief review out-lines recent advances using nanoparticles in bioanalyticswith emphasis on small metal colloids and semiconductor quantum dots.

Nanoparticles are primarily being introduced into bio-analytics as an improved alternative to conventional fluo-rescent labels or label-free sensing elements, such as re-placements for organic dyes or metallic thin layers used inSurface Plasmon Resonance (SPR). Like metallic thin

films, Au and Ag nanoparticles display a Plasmon Reso-nance that is sensitive to refractive index changes in thesurrounding environment. However, unlike metallic thinfilms that display a polarization-dependent infrared Plas-mon Resonance, because of confinement effects, nanopar-ticles display a Localized Surface Plasmon Resonance(LSPR) displaying an absorption peak in the visible spec-trum and excitable with non-polarized light. In addition,these nanoparticles produce intense Surface EnhancedRaman Spectra (SERS) effects. Semiconductor nanoparti-cles, commonly called quantum dots, have dimensionssmaller than the Bohr radius of an electron in the bulk ma-terial. This confines the electron/hole pairs such that be-

haviour mimics a particle in a box. The most popular quantum dots have large fluorescence quantum yields, re-sistance to photobleaching, good chemical stability, andGaussian emission profiles (~30nm FWHM). One cantune the properties of nanoparticles by controlling the

size, shape, and preparation procedures. For example, theemission from CdSe quantum dots can be adjusted nearlycontinuously from 450–650nm. Nanoparticle sizes, typi-cally on the order of 10nm, are comparable to biologicalproteins. Nanoparticles can be suspended in solution for in-vivo studies or aggregated in certain conditions, thusproviding a simple separation tool. Figure 1 is an exampleof the brilliant images that are possible when biologicalsamples are labelled with CdSe quantum dots. Several re-cent reviews discuss the properties of nanoparticles ingreater detail [1, 2, 3].

To date, the stability and solubility in aqueous solu-tions is the largest limiting factor that keeps nanoparticlesfrom being pervasive in bioanalytical laboratories. To bebiologically relevant, nanoparticles need to have surface

Dale M. Willard

Nanoparticles in bioanalytics

Anal Bioanal Chem (2003) 376: 284–286DOI 10.1007/s00216-002-1734-8

Published online: 15 February 2003

TRENDS

D. M. Willard (✉)Institute for Physical Chemistry, University of Tuebingen,Auf der Morgenstelle 8, 72076 Tuebingen, Germanye-mail: [email protected]

© Springer-Verlag 2003

Fig.1 Microtubules in 3T3 cells labelled with Qdot 605 Strepta-vidin Conjugate after the cells were incubated with monoclonalanti-tubulin antibodies and biotinylated goat anti-mouse IgG. Nu-clei were counter-stained with blue dye Hoechst. Image was cap-tured with a Nikon E-800 fluorescent microscope equipped with aCCD camera. Image courtesy of Quantum Dot Corporation, Hay-ward, CA, USA. www.qdots.com



functionality amenable to biological derivatisation, solu-

bility and long term stability in a range of buffered salinesolutions and pHs, and limited non-specific binding. Manyrecent studies have addressed these issues. The Alivasatosgroup developed CdSe quantum dots coated with siloxaneand poly(ethylene glycol) (PEG) layers [4, 5]. Thesequantum dots were derivatised with DNA that was thenused to capture the quantum dots on DNA labelled sur-faces. Mattoussi and coworkers developed a novel elec-trostatic coupling of quantum dots and proteins [6, 7, 8].Others have developed new thiolyting groups to stabilisethe surface chemistry [9, 10]. Another study found that Agnanoparticles can be stabilised by adding a thin layer of Au without disrupting the underlying Ag LSPR property

[11].Two papers developed different quantum dot derivati-sation procedures that suppress non-specific binding andtargets them to specific sites within biological samples.The first study demonstrated fluorescence in-situ hybrid-ization experiments[9]. Quantum dots were derivatised

with a hydroxyl-disulfide organic cap to reduce non-spe-cific binding and further derivatised with oligonucleotidesthat targeted them to chromosomes in human sperm cells.A more recent study found that quantum dots derivatisedwith PEG and polypeptides can be used for in-vivo stud-ies [12]. The PEG reduced non-specific binding and thepeptides directed the quantum dots to the lungs of mice.

Since quantum dots were first demonstrated as biolog-

ical probes [13, 14], they have been promoted for multi-label experiments. Different sets of nanoparticles can besimultaneously excited with a single laser source, de-tected individually by their unique emission/scattering,and biologically derivatised with similar procedures (seeFig. 2). Recently, Mattoussi and co-workers demonstrateda dual-quantum dot assay [6]. Other papers utilized quan-tum dots in Energy-Transfer studies [15, 16]. Mirkin andco-workers demonstrated a dual-Au nanoparticle assaywith detection via scattering [17]. In addition, two papersfound Au nanoparticles to be very efficient fluorescencequenchers and can be effectively utilized in Molecular Beacon or Molecular Beacon-like assays[18, 19].

Nanoparticles hold particular promise as the next gen-eration barcodes for multiplexing experiments (see Fig. 3).Genomic and proteomic research demands greater infor-mation from single experiments. Conventional experi-ments utilize multiple organic fluorophores to barcodedifferent analytes in a single experiment, but positiveidentification is difficult because of the cross-talk signalbetween fluorophores. Han et al. encapsulated CdSe quan-tum dots in polystyrene beads with varying concentrationsand sizes [20]. The narrow Gaussian emission profile of quantum dots reduces cross-talk. Therefore, eight beadvariations were clearly identifiable with many more possi-ble. Another study took advantage of the large SERS ef-

fect next to Au nanoparticles plated with Ag [21]. Nano-particles labelled with dyes produced a clear fingerprintrevealed by the Raman Spectra. Another study preparednanometer-micrometer length metal rods coded withstrips of differing composition that could be identified op-tically via reflectivity [22]. Yet another study found that

285

Fig.2 A family of quantum dots emits a spectrum of colourswhen excited with a single light source. Image copyrighted: FeliceFrankel, Envisioning Science, the Design and Craft of the ScienceImage

Fig.3 Graphic depicting howquantum dots can be insertedinto polystyrene beads to cre-ate distinct spectral barcodes.Image courtesy of QuantumDot Corporation, Hayward,CA, USA. www.qdots.com



286

spherical Ag nanoparticles can be converted to triangular prisms with simple UV irradiation, adding shape as a dis-tinguishing feature of mixed nanoparticles in solution[23].

Several studies demonstrated Au [24, 25] or Ag[26,27] functionalised colloids/nanoparticles can be utilisedas an alternative to SPR. The assays are label-free withsurface binding events inducing a shift in the LSPR. As

opposed to conventional SPR, changes in the LSPR canbe monitored with simple UV–visible spectrometers andthe technology is easily transferred to high density arrayplatforms. However to date, nanoparticles based assayscan not match the limit of detection obtained by conven-tional SPR.

Functionalised nanoparticles dispersed in solution canbe aggregated by adding a cross-linking reagent. TheLSPR is very sensitive to aggregation and can be moni-tored with UV–visible spectrometry. In addition, aggre-gates can be separated from other solvated components bycentrifugation. Three papers utilized these advantages todevelop assays for lectin[28], anti-Protein A antibodies

[29], and metal ions [30]. Another study extended theseadvantages by labelling the nanoparticles with oligonu-cleotide-recognition elements [31]. This permitted multi-analyte assays since separated aggregates could then beidentified by their oligonucleotide-elements with either thermal de-hybridisation or chip-based detection.

Three final, but no less noteworthy articles can beviewed as unique research. Hamad-Schifferli et al. [32]remotely heated Au functionalised nanoparticles by a ra-dio frequency magnetic field. The local heating of thenanoparticles induced DNA de-hybridisation that couldthen be re-hybridised when the field was removed, in ef-fect producing electronically controlled nano-”machin-

ery”. Park et al. [33] developed a very sensitive DNA as-say detected with simple electrical conductance measure-ments. They developed methods to grow Ag wires be-tween micro-gates containing Au nanoparticles. The Aunanoparticles were functionalised with oligonucleotidesthat permitted binding to the gate region of the chip onlywhen target analytes are present; 500fmol L –1 concentra-tions were detected. The authors propose that the assay iseasily amenable to micro-array formats. Finally, Gearhertet al. [34] used Au nanoparticles as a template for thestudy of DNA curvature conformations. Utilizing SERSeffects, they measured oligonucleotides breathing vibra-tions to identify the presence of straight, bent, or kinkedDNA.

Clearly, nanoparticles have a promising future in bio-analytics. Their utilisation will be driven by the need for smaller detection platforms with lower limits of detection.Once nanoparticles become commercially available andtheir solubility and stability issues addressed, we can ex-pect great things from these little packages.

References

1. Chan WCW, Maxwell DJ, Gao XH, Bailey RE, Han MY, NieSM (2002) Curr Opin Biotech 13:40–46

2. Murphy CJ, Coffer JL (2002) Appl Spectrosc 56:16A–26A3. Klarreich E (2001) Nature 413:450–4524. Gerion DP, Wolfgang J, Williams SC, Zanchet D, Micheel

CM, Alivisatos AP (2001) J Am Chem Soc 124:7070–70745. Parak WJ, Gerion D, Zanchet D, Woerz AS, Pellegrino T,

Micheel C, Williams SC, Seitz M, Bruehl RE, Bryant Z, Bus-tamante C, Bertozzi CR, Alivisatos AP (2002) Chem Mater 14:2113–2119

6. Goldman ER, Balighian ED, Mattoussi H, Kuno MK, MauroJM, Tran PT, Anderson GP (2002) J Am Chem Soc 124:6378–6382

7. Goldman ER, Balighian ED, Kuno MK, Labrenz S, Tran PT,Anderson GP, Mauro JM, Mattoussi H (2002) Phys Stat Sol B229:407–414

8. Goldman ER, Andersen GP, Tran PT, Mattoussi H, CharlesPT, Mauro JM (2002) Anal Chem 74:841–847

9. Pathak S, Choi SK, Armheim N, Thompson ME (2001) J AmChem Soc 123:4103–4104

10. Li Z, Jin R, Mirkin CA, Letsinger RL (2002) Nucleic AcidsRes 30:1558–1562

11. Cao YW, Jin R, Mirkin, CA (2001) J Am Chem Soc 123:

7961–796212. Akerman ME, Chan WCW, Laakkonen P, Bhatia SN, Rous-

lahti E (2002) Proc Nat Aca Sci 99:12617–1262113. Bruchez MJ, Moronne M, Gin P, Weiss S, Alivisatos AP

(1998) Science 281:2013–201614. Chan WCW, Nie S (1998) Science 281:2016–201815. Wang S, Mamedova N, Kotov NA, Chen W, Studer J (2002)

Nano Lett 2:817–82216. Willard DM, Carillo LL, Jung J, Van Orden A (2001) Nano

Lett 1:469–47417. Taton TA, Lu G, Mirkin CA (2001) J Am Chem Soc 123:

5164–516518. Dubertret B, Calame M, Libchaber AJ (2001) Nature Biotech

19:365–37019. Maxwell DJ, Taylor JR, Nie S (2002) J Am Chem Soc 124:

9606–9612

20. Han M, Gao X, Su JZ, Nie S (2001) Nature Biotech 19:631– 635

21. Cao YC, Jin R, Mirkin CA (2002) Science 297:1536–154022. Nicewarner-Pena SR, Freeman RG, Reiss BD, He L, Pena DJ,

Walton ID, Cromer R, Keating CD, Natan MJ (2001) Science294:137–141

23. Jin R, Cao Y, Mirkin CA, Kelly KL, Schatz GC, Zheng JG(2001) Science 294:1901–1903

24. Kalyuzhny G, Schneeweiss MA, Shanzer A, Vaskevich A,Rubinstein I (2001) J Am Chem Soc 123:3177–3178

25. Nath N, Chilkoti A (2002) Anal Chem 74:504–50926. Malinsky MD, Kelly KL, Schatz GC, Van Duyne RP (2001)

J Am Chem Soc 123:1471–148227. Haes AJ, Van Duyne RP (2002) J Am Chem Soc 124:10596–

1060428. Otsuka H, Akiyama Y, Nagasaki Y, Kataoka K (2001) J Am

Chem Soc 123:8226–823029. Thanh NTK, Rosenzweig Z (2002) Anal Chem 74:1624–162830. Kim Y, Johnson RC, Hupp JT (2001) Nano Lett 1:165–16731. Nam JM, Park SJ, Mirkin CA (2002) J Am Chem Soc 124:

3820–382132. Hamad-Schifferli K, Schwartz JJ, Santos AT, Zhang S, Jacob-

son JM (2002) Nature 415:152–15533. Park SJ, Taton TA, Mirkin CA (2002) Science 295:1503–150634. Gearheart LA, Ploehn HJ, Murphy CJ (2001) J Phys Chem B

105:12609–12615

dale m. willard- nanoparticles in bioanalytics

Documents