Nanoparticle-based biosensors and bioassays - Electrochemical Sensors, Biosensors and Their Biomedical Applications - page 444

see Plate 14 for color version). Magnetic separation of the complexed probes and
target, followed by thermal dehybridization of the oligonucleotides on the nanopar-
ticle surface, allowed highly sensitive scanometric measurements of the target pro-
tein by identifying the oligonucleotide sequence released from the nanoparticle
probe. Substantial amplifi cation was achieved because each nanoparticle probe car-
ried a large number of oligonucleotides per protein binding event. A multianalyte
immunoassay could be accomplished by using different oligonucleotide sequences
for encoding different target antigens. The nanoparticle-based bio-barcode assay
was applied for detecting the prostate-specifi c antigen (PSA) at the low attomolar
(aM 10 18 M) level, that is, a sensitivity that is six orders of magnitude greater than

the corresponding ELISA assay. In addition, the same assay was used for measur-
ing the pathogenic Alzheimer's disease marker ADDL in the cerebral spinal fl uid at
picomolar levels [18]. The bio-barcode concept was extended also to PCR-less ampli-
fi ed detection of DNA hybridization down to the 500 zeptomolar level (i.e. 30 cop-
ies in 30 10 21 M) [19]. A recent contribution from Niemeyer and

coworkers demonstrated a sensitive optical detection of proteins using difunctional
DNA-gold nanoparticles [20]. In this case, the sandwich immunoassay caused mul-
tilayers of DNA-linked gold nanoparticles to form that were detected by ultraviolet/
visible (UV/Vis) spectroscopy.
Another attractive route for optical transduction of biorecognition events involves
the encapsulation of a huge amount of a fl uorescent marker within nanoparticle carriers
[21-26]. Harma et al. reported a europium-entrapped fl uorescence nanoparticle label
for an ultrasensitive prostate-specifi c antigen (PSA) assay. PSA was detected in micro-
titer wells coated with a PSA-specifi c antibody using biotinylated antibody and strepta-
vidin-coated, highly fl uorescent 107 nm nanoparticles that contained more than 30 000
europium ions entrapped by beta-diketones. PSA was monitored directly on the surface
of a well without any additional enhancement step. The sensitivity of the assay was
1.6 ng/L, corresponding to 50 fmol L 1 fM or 250 zeptomoles (250

µ L samples; zM

10 21 mol 1 )
of PSA. The high specifi c activity and low non-specifi c binding of the streptavidin
coated nanoparticles improved the sensitivity of the PSA assay 100-fold compared to
the conventional europium-labeled streptavidin tracer in the same assay format. This
nanoparticle label has been successfully used for free PSA detection in a serum sample
[22] and thyroid-stimulating hormone (a marker of thyroid function) detection [23].
Trau and coworkers reported on a highly sensitive immunoassay of proteins based
on polyelectrolyte encapsulated microcrystalline fl uorescent material interfaced to
the antibody [24]. A dramatically ( 2000-fold) amplifi ed immunoassay was reported

in connection with the release of the fl uorescent molecules to the detection medium
following the antibody-antigen interaction. An analogous route for maximizing the
number of fl uorescent molecules per binding event was reported by Tan and coworkers
[25]. For this purpose, the fl uorescent dye was encapsulated within silica nanoparticles
functionalized with an oligonucleotide probe. The method offered an extremely low
detection limit of 0.8 fM with effective discrimination against mismatched DNA. The
silica matrix also provided good protection against bleaching of the fl uorophore. The
fl uorescence-bioconjugated silica nanoparticles were also applied for ultrasensitive
bacterial detection, down to a single E. coli O157 cell [26].