Nonenzymatic detection of bacterial genomic DNA using the bio bar code assay.
ABSTRACT The detection of bacterial genomic DNA through a nonenzymatic nanomaterials-based amplification method, the bio bar code assay, is reported. The assay utilizes oligonucleotide-functionalized magnetic microparticles to capture the target of interest from the sample. A critical step in the new assay involves the use of blocking oligonucleotides during heat denaturation of the double-stranded DNA. These blockers bind to specific regions of the target DNA upon cooling and prevent the duplex DNA from rehybridizing, which allows the particle probes to bind. Following target isolation using the magnetic particles, oligonucleotide-functionalized gold nanoparticles act as target recognition agents. The oligonucleotides on the nanoparticle (bar codes) act as amplification surrogates. The bar codes are then detected using the Scanometric method. The limit of detection for this assay was determined to be 2.5 fM, and this is the first demonstration of a bar code-type assay for the detection of double-stranded, genomic DNA.
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ABSTRACT: Prussian blue nanoparticles (PBNPs) have been certified a kind of mimetic enzyme possesses the advantages of stability, high catalytic activity and low prices. Ferritins are natural nanoscale structures with unique three-dimensional structures and biological functions. In this context, we synthesized PBNPs on the surface of the iron oxide core of ferritin taking use of the ferric iron of the core, we also intended to retain the specificity of ferritin for some biological use. Our results show the resulting nanostructures (Prussian blue-ferritin nanoparticles, PB-Ft NPs) got very small size and relatively high catalytic activity , furthermore, PB-Ft NPs successfully combined the intrinsic enzyme mimetic activity of PBNPs and the specificity to tumor cells of ferritin. The peroxidase-like activity and catalase-like activity of PB-Ft NPs were studied. Peroxidase-like activity which fits well the Michaelis-Menten kinetics was found strongly depending on pH, temperature and the concentration of PB-Ft NPs. Then a sensitive method for glucose detection was developed using glucose oxidase (GOx) and PB-Ft NPs. PB-Ft NPs displayed catalase-like activity in PH higher than 5.0, the generated oxygen was measured by the dissolved oxygen electrode.Enzyme-linked immunosorbent assay (ELISA) shows PB-Ft NPs possess both specificity and peroxidase-like activity.Key Engineering Materials 07/2013; 562-565:1333-1339.
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ABSTRACT: The analysis of ancient or processed DNA samples is often a great challenge, because traditional Polymerase Chain Reaction - based amplification is impeded by DNA damage. Blocking lesions such as abasic sites are known to block the bypass of DNA polymerases, thus stopping primer elongation. In the present work, we applied the SERRS-hybridization assay, a fully non-enzymatic method, to the detection of DNA refractory to PCR amplification. This method combines specific hybridization with detection by Surface Enhanced Resonant Raman Scattering (SERRS). It allows the detection of a series of double-stranded DNA molecules containing a varying number of abasic sites on both strands, when PCR failed to detect the most degraded sequences. Our SERRS approach can quickly detect DNA molecules without any need for DNA repair. This assay could be applied as a pre-requisite analysis prior to enzymatic reparation or amplification. A whole new set of samples, both forensic and archaeological, could then deliver information that was not yet available due to a high degree of DNA damage.PLoS ONE 01/2014; 9(12):e114148. · 3.53 Impact Factor
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ABSTRACT: A simple and ultrasensitive detection of human IgG based on signal amplification using a novel bio-barcode assay and DNA chip technology was developed. The sensing platform was a sandwich system made up of antibody-modified magnetic microparticles (Ab-MMPs)/human IgG/Cy3-labeled single-stranded DNA and antibody-modified gold nanoparticles (Cy3-ssDNA-Ab-AuNPs). The MMPs (2.5 μm in diameter) modified with mouse anti-human IgG monoclonal-antibodies could capture human IgG and further be separated and enriched via a magnetic field. The AuNPs (13 nm in diameter) conjugated with goat anti-human IgG polyclonal-antibodies and Cy3-ssDNA could further combine with the human IgG/Ab-MMP complex. The Cy3-ssDNA on AuNPs was then released by TCEP to hybridize with the DNA chip, thus generating a detectable signal by the fluorescence intensity of Cy3. In order to improve detection sensitivity, a three-level cascaded signal amplification was developed: (1) The MMP enrichment as the first-level; (2) Large quantities of Cy3-ssDNA on AuNPs as the second-level; (3) The Cy3-ssDNA conjugate with DNA chip as the third-level. The highly sensitive technique showed an increased response of the fluorescence intensity to the increased concentration of human IgG through a detection range from 1 pg mL−1 to 10 ng mL−1. This sensing technique could not only improve the detection sensitivity for the low concentration of human IgG but also present a robust and efficient signal amplification model. The detection method has good stability, specificity, and reproducibility and could be applied in the detection of human IgG in the real samples.Journal of Nanoparticle Research 09/2013; 15(9). · 2.28 Impact Factor
Nonenzymatic Detection of Bacterial Genomic
DNA Using the Bio Bar Code Assay
Haley D. Hill, Rafael A. Vega, and Chad A. Mirkin*
Department of Chemistry and International Institute for Nanotechnology, Northwestern University, 2145 Sheridan Road,
Evanston, Illinois 60208-3113
The detection of bacterial genomic DNA through a non-
enzymatic nanomaterials-based amplification method, the
bio bar code assay, is reported. The assay utilizes oligo-
nucleotide-functionalized magnetic microparticles to cap-
ture the target of interest from the sample. A critical step
in the new assay involves the use of blocking oligonucle-
otides during heat denaturation of the double-stranded
DNA. These blockers bind to specific regions of the target
DNA upon cooling and prevent the duplex DNA from
rehybridizing, which allows the particle probes to bind.
Following target isolation using the magnetic particles,
oligonucleotide-functionalized gold nanoparticles act as
target recognition agents. The oligonucleotides on the
nanoparticle (bar codes) act as amplification surrogates.
The bar codes are then detected using the Scanometric
method. The limit of detection for this assay was deter-
mined to be 2.5 fM, and this is the first demonstration of
a bar code-type assay for the detection of double-stranded,
Polymerase chain reaction (PCR)-based amplification tech-
niques1-3have become standard methodologies for the detection
of nucleic acids.4,5With the advent of quantitative real time PCR
and variants of it such as reverse transcription PCR, one can now
detect nucleic acid targets in a highly quantitative manner and
assess important processes like gene expression.6-10Though there
are many benefits to PCR such as sensitivity, production of a
usable product fragment, and the ability to sequence that frag-
ment, there are times when these features of PCR are not
necessary and the cumbersome nature of PCR is a disadvantage.
For example, in the case of point-of-care biological detection
applications, where speed is critical and the enzymatic constraints
and cost of PCR are limiting,11an enzyme-free approach could be
a major advantage.
The recently developed bio bar code assay for the detection
of protein and nucleic acid targets is potentially capable of filling
this void. This assay has several forms12-16and has shown promise
in the high-sensitivity detection of protein and oligonucleotide
targets. In addition, it has the ability to simultaneously detect many
different targets in one sample.17-19Recently it has been adapted
to a microfluidic chip-based format, an important step toward
automation.20With respect to nucleic acids, thus far, all proof-of-
concept work has involved short nucleic acids in very clean
environments (buffer). The complexities of the target and sample
media are often limiting factors for any nucleic acid assay,
especially ones that rely on enzymes for amplification. This made
us wonder if the bio bar code assay could overcome such
limitations. Herein, we describe the development of a new version
of the bio bar code assay that utilizes blocking strands to inhibit
target rehybridization and allows one to detect double stranded
genomic DNA at a limit of detection (LOD) of 2.5 fM (7.5 × 104
copies/50 µL). Proof-of-concept studies in the context of Bacillus
subtilis DNA detection are reported.
Gold nanoparticles heavily functionalized with oligonucleotides
(oligo-AuNPs) are the cornerstone of the bio bar code assay.21
These oligo-AuNPs have a variety of attributes with respect to
* To whom correspondence should be addressed. E-mail: chadnano@
(1) Saiki, R. K.; Scharf, S.; Faloona, F.; Mullis, K. B.; Horn, G. T.; Erlich, H. A.;
Arnheim, N. Science 1985, 230, 1350-1354.
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Harbor Symp. Quant. Biol. 1986, 51, 263-273.
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(5) Jochen-Wilhelm, A. P. ChemBioChem 2003, 4, 1120-1128.
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Harbor Press: Cold Spring Harbor, NY, 2003.
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(10) Higuchi, R.; Fockler, C.; Dollinger, G.; Watson, R. Bio-Technology 1993,
(11) Mirkin, C. A.; Thaxton, C. S.; Rosi, N. L. Expt. Rev. Mol. Diagn. 2004, 4,
(12) Nam, J. M.; Park, S. J.; Mirkin, C. A. J. Am. Chem. Soc. 2002, 124, 3820-
(13) Nam, J. M.; Stoeva, S. I.; Mirkin, C. A. J. Am. Chem. Soc. 2004, 126, 5932-
(14) Thaxton, C. S.; Hill, H. D.; Georganopoulou, D. G.; Stoeva, S. I.; Mirkin, C.
A. Anal. Chem. 2005, 77, 8174-8178.
(15) Oh, B. K.; Nam, J. M.; Lee, S. W.; Mirkin, C. A. Small 2006, 2, 103-108.
(16) Bao, Y. P.; Wei, T. F.; Lefebvre, P. A.; An, H.; He, L. X.; Kunkel, G. T.;
Muller, U. R. Anal. Chem. 2006, 78, 2055-2059.
(17) Stoeva, S. I.; Lee, J. S.; Smith, J. E.; Rosen, S. T.; Mirkin, C. A. J. Am. Chem.
Soc. 2006, 128, 8378-8379.
(18) Stoeva, S. I.; Lee, J. S.; Thaxton, C. S.; Mirkin, C. A. Angew. Chem., Int. Ed.
2006, 45, 3303-3306.
(19) Georganopoulou, D. G.; Chang, L.; Nam, J. M.; Thaxton, C. S.; Mufson, E.
J.; Klein, W. L.; Mirkin, C. A. P. Natl. Acad. Sci. U.S.A. 2005, 102, 2273-
(20) Goluch, E. D.; Nam, J. M.; Georganopoulou, D. G.; Chiesl, T. N.; Shaikh,
K. A.; Ryu, K. S.; Barron, A. E.; Mirkin, C. A.; Liu, C. Lab Chip 2006, 6,
(21) (a) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. Nature 1996,
382, 607-609. (b) Letsinger, R. L.; Elghanian, R.; Viswanadham, G.; Mirkin,
C. A. Bioconj. Chem. 2000, 11, 289-291. (c) Li, Z.; Jin, R.; Mirkin, C. A.;
Letsinger, R. L. Nucleic Acids Res. 2002, 30, 1558-1562.
Anal. Chem. 2007, 79, 9218-9223
Analytical Chemistry, Vol. 79, No. 23, December 1, 2007
10.1021/ac701626y CCC: $37.00© 2007 American Chemical Society
Published on Web 10/10/2007
probe design. They are easily functionalized,21highly tailorable,22,23
remarkably stable,24catalytic,25and cooperative binders (they
exhibit unusually sharp melting transitions when hybridized to
complementary DNA).26These sharp melting transitions can
confer a considerable selectivity advantage to the oligo-AuNPs
over their PCR primer counterparts.27Oligo-AuNPs serving as
amplification agents in the bio bar code assay, through the
chemical release of their oligonucleotide “bar codes”, have several
potential advantages over Taq-polymerase or other DNA replica-
tion enzymes. For example, the oligo-AuNP probes are stable for
extended periods (greater than 6 months) at ambient tempera-
ture21and are resistant to degradation,28while polymerases, like
most enzymes, need to be stored at 4 °C or below. Oligo-AuNPs
also function in a host of complex conditions such as sodium
chloride concentrations up to 1 M,29different buffers such as Tris
(2-amino-2-(hydroxymethyl)-1,3-propanediol), phosphate (Na2-
HPO4, NaH2PO4), borate (Na2B4O7), and MOPS (3-(N-morpholi-
no)propanesulfonic acid) and in the presence of metal ions or
small molecules without adverse effects on their activity.30-37
The bio bar code assay combines target-specific oligo-AuNPs
with a second homogeneous capture agent (a magnetic micro-
particle functionalized with a different target-specific oligonucle-
otide, oligo-MMP). The oligo-MMP is used to capture and isolate
the target of interest from the sample solution, prior to the addition
of the oligo-AuNPs. The MMP-target-AuNP complex allows for
rapid isolation and subsequent washing prior to oligonucleotide
bar code release. The bar code can be easily detected via the
Scanometric method, which exhibits an LOD for short purified
oligonucleotides of 100 aM.25,38
MATERIALS AND METHODS
Culture Media and Bacterial Strains. Routine growth and
maintenance of B. subtilis 168 (American Type Culture Collection
23857) was done in Luria-Bertani (LB) media (Fisher Scientific)
and on solidified plates using LB agar (Fisher Scientific). All
cultures were maintained at 30 °C on plates or in liquid form with
shaking at 160 rpm and 30 °C. All growth media were sterilized
by autoclave treatment prior to use.
Genomic DNA Isolation. B. subtilis cells were grown in 50
mL of liquid media in 125-mL flasks overnight and harvested after
10 h of growth. The cells were split into two 25-mL aliquots and
spun down at 8000 rpm for 10 min. The supernatant was then
removed, and the aliquots were resuspended in 5 mL of 50 mM
Tris, 50 mM EDTA at pH 8.0 and frozen for no less than 1 h at
-20 °C. Frozen cells in a 50-mL conical tube were placed on ice,
and 500 µL of 10 mg/mL lysozyme (Fisher Scientific) dissolved
in 250 mM Tris, pH 8.2 was added to the tube. The cells-lysozyme
mixture was allowed to slowly warm to room temperature over a
2-h period. Next, 1 mL of a 1 mg/mL solution of proteinase K
(Fisher Scientific) in 50 mM Tris, 0.4 M ETDA, 0.5 M SDS, pH
7.5 was incubated with the cells at 50 °C for 1 h. Afterward, RNase
A (1 µL, Ambion Inc./Applied Biosystems) was added to degrade
all RNA contamination. Following RNA degradation, the genomic
DNA was removed from the other cellular debris by phenol-
chloroform extraction and ethanol precipitation. The integrity and
size of the genomic DNA was confirmed by gel electrophoresis
using a 1% agarose gel with ethidium bromide (Bio-Rad, ReadyA-
garose Gels) with 1× TBE (Tris, boric acid, EDTA) buffer at 120
V for 1 h. (Supporting Information Figure S1, lane 5/6) The size
of the genomic DNA isolated was compared with commercially
available genomic DNA isolated by ATCC for B. subtilis 168.
(Figure S1, lane 2).
Probe Design. Probes were designed from the R subunit of
the tryptophan synthase gene (bp 2371552-2370749) from B.
subtilis 168. All probes were tested against the NCBI BLAST
search engine (http://www.ncbi.nlm.nih.gov/BLAST/), with the
magnetic and gold probe sequences being unique to B. subtilis.
Two of the three blocking sequences (center and 5′) had a
homology to one other organism, while the 3′ blocker was specific
only for B. subtilis. Probes were designed to fall within a region
of the genome that could be cut easily with the restriction enzyme
HpyCH4V (New England Biolabs). This was done to allow for
decircularization of the genomic DNA and prevention of super-
coiling during the heat denaturation step of the assay. Probe
specificity was confirmed by routine Southern blot analysis (data
not shown). Oligonucleotide sequences are given in Table 1.
Oligonucleotides. All specialty oligonucleotides were pur-
chased from Integrated DNA Technologies (Coralville, IA) and
were purified by HPLC. Standard desalting was used to purify
PCR primers and blocking strands. Prior to use, the oligonucle-
otides were stored at-80 °C in a dried state. Working solutions of
the oligonucleotides were stored at-20 °C.
Quantitative PCR. The copy number of genomic DNA per
milliliter (isolated from B. subtilis cells) was determined using
quantitative real-time PCR (qPCR). A LightCycler 2.0 instrument
and LightCycler Software Version 4.0 (Roche Applied Sciences)
were used to run the qPCR reactions and quantify the data,
respectively. Primers were designed to amplify a 1066-base pair
fragment of the genomic DNA from B. subtilis (Table 1). A
LightCycler FastStart DNA Master SYBR Green kit (Roche
Applied Sciences) was used to generate the PCR reactions. Each
PCR reaction was carried out in a 20-µL capillary (Roche Scientific)
by placing the capillary tube in a cooling rack (4 °C), combining
and mixing the reagents in an eppendorf tube at 4 °C, spinning
the reactants into the capillary tubes, and thermally cycling. Since
(22) Li, Z.; Jin, R.; Mirkin, C. A.; Letsinger, R. L. Nucleic Acids Res. 2002, 30,
(23) Cao, Y. W. C.; Jin, R.; Mirkin, C. A. Science 2002, 297, 1536-1540.
(24) Storhoff, J. J.; Lazarides, A. A.; Mucic, R. C.; Mirkin, C. A.; Letsinger, R. L.;
Schatz, G. C. J. Am. Chem. Soc. 2000, 122, 4640-4650.
(25) Taton, T. A.; Mirkin, C. A.; Letsinger, R. L. Science 2000, 289, 1757-
(26) Jin, R.; Wu, G. S.; Li, Z.; Mirkin, C. A.; Schatz, G. C. J. Am. Chem. Soc.
2003, 125, 1643-1654.
(27) Lytton-Jean, A. K. R.; Mirkin, C. A. J. Am. Chem. Soc. 2005, 127, 12754-
(28) Rosi, N. L.; Giljohann, D. A.; Thaxton, C. S.; Lytton-Jean, A. K. R.; Han, M.
S.; Mirkin, C. A. Science 2006, 312, 1027-1030.
(29) Hurst, S. J.; Lytton-Jean, A. K. R.; Mirkin, C. A. Anal. Chem. 2006, 78,
(30) Han, M. S.; Lytton-Jean, A. K. R.; Oh, B. K.; Heo, J.; Mirkin, C. A. Angew.
Chem., Int. Ed. 2006, 45, 1807-1810.
(31) Han, M. S.; Lytton-Jean, A. K. R.; Mirkin, C. A. J. Am. Chem. Soc. 2006,
(32) Lee, J-S; Han, M. S.; Mirkin, C. A. Angew. Chem., Int. Ed. 2007, 46, 4093-
(33) Liu, J. W.; Lu, Y. J. Am. Chem. Soc. 2003, 125, 6642-6643.
(34) Liu, J. W.; Lu, Y. Anal. Chem. 2004, 76, 1627-1632.
(35) Daniel, M. C.; Astruc, D. Chem. Rev. 2004, 104, 293-346.
(36) He, L.; Musick, M. D.; Nicewarner, S. R.; Salinas, F. G.; Benkovic, S. J.;
Natan, M. J.; Keating, C. D. J. Am. Chem. Soc. 2000, 122, 9071-9077.
(37) Huang, C. C.; Huang, Y. F.; Cao, Z. H.; Tan, W. H.; Chang, H. T. Anal.
Chem. 2005, 77, 5735-5741.
(38) Storhoff, J. J.; Sudhakar S.; Garimella, Viswanadham; Mirkin, Chad A. In
Microarray Technology and Its Applications; Mueller, U. R. N., Dan V., Ed.;
Springer GmbH: Berlin, Germany, 2005,; pp 147-179.
Analytical Chemistry, Vol. 79, No. 23, December 1, 2007
the real-time reporter used (SYBR Green) is an intercalating
fluorophore where total fluorescence depends on product length,
a standard curve for the qPCR had be constructed beforehand.
To do this, end-point PCR was run using Qiagen’s Taq PCR Master
Mix following the manufacturer’s protocol. During each amplifica-
tion, primer concentrations of 0.5 µM were used with ∼0.1 µg of
template. The reactions were done in 100-µL PCR tubes in strips
of eight (Fisher Scientific) on a GeneAmp PCR System (Applied
BioSystems). The thermal profile consisted of an initial 8-min
denaturation step at 95 °C, followed by 45 cycles of denaturation
at 95 °C for 55 s, annealing at 52 °C for 45 s, and extension at
72 °C for 120 s. Following the 45 cycles, the product sequence
was completed with an extension step at 72 °C for an additional
10 min. The PCR product size was confirmed by gel electrophore-
sis using a 1% agarose gel with ethidium bromide (Bio-Rad,
ReadyAgarose Gels) with 1× TBE buffer at 120 V for 1 h (Figure
S1, lane 3/4). The remaining PCR product, not run on the gel,
was purified using a MinElute PCR purification kit (Qiagen Inc.)
following the manufacturer’s instructions. The PCR product was
quantified using UV-visible spectroscopy, and then was used to
generate a standard curve for qPCR (Supporting Information
Magnetic Particle Functionalization with DNA. Oligonucle-
otide-functionalized magnetic microparticles (oligo-MMPs) were
prepared according to literature procedures.17,39Briefly, 2.8-µm
amine-functionalized magnetic microparticles (Dynal Corp./Invit-
rogen) were coupled to thiolated oligonucleotide strands using
the heterobifunctional cross-linker sulfo-SMPB (Pierce Biotech-
nology). Unreacted amine sites were passivated with sulfo-NHS
acetate (Pierce Biotechnology). Oligo-MMPs were stored at 4 °C
in 10 mM phosphate-buffered saline (0.15 M NaCl; PBS) with
0.001% sodium azide as a preservative. Oligo-MMPs were washed
three times with PBS prior to use in the assay.
Gold Nanoparticle Functionalization with DNA. Oligo-
AuNP probes were prepared according to literature procedures.39
Briefly, 4 nmol of freshly reduced thiolated DNA was added to 1
mL of 13 ( 2-nm gold nanoparticles and shaken gently overnight.
The system was buffered to a phosphate concentration of 10 mM
(pH 7.0) including 0.01% sodium dodecyl sulfate (SDS). Over the
course of 1 day, the sodium chloride concentration was brought
to 0.15 M in a stepwise manner. Particles were then spun (13 000
rpm) and rinsed four times (10 mM phosphate, 0.15 M NaCl, 0.01%
SDS, pH 7.4) to remove any unbound DNA. Probes were stored
in excess DNA until needed, at which time they were purified as
Melting Analysis. In order to determine the maximum assay
stringency, or the upper temperature limit to heat denature and
eliminate nonspecific hybridization events in solution, we deter-
mined the melting temperatures (Tm) for each of the probes (oligo-
AuNP and oligo-MMP probe sequences) with their respective
targets in a 1:1 ratio (eq 1).
In a typical melting experiment, AuNPs were functionalized with
a 5′-thiol-modified probe sequence (Table 1) and allowed to hybrid-
ize to 1 equivalent of a 5′-fluorescein (FITC)-modified comple-
mentary DNA sequence specific to the targeted regions within
the tryptophan synthase gene (trp 1; AuNP target, trp 3; MMP
All experiments were allowed to equilibrate for over 24 h in
10 mM phosphate buffer, 0.15 M NaCl, 0.1% SDS, pH 7.4 (assay
buffer) to ensure that equilibrium has been reached. Binding of
the nanoparticle probes to a complementary target sequence
modified with a molecular fluorophore resulted in quenching and
decreased fluorescence intensity. Subsequent heating resulted in
dissociation of the probe-target complex and a recovery of
fluorescence intensity, providing a way to spectroscopically moni-
tor the melting transition (Figure 1). Fluorescence measurements
were performed on a Molecular Devices Gemini EM Microplate
spectrofluorometer with temperature control. Comparison of the
trp 1 (Au NP) probe-target (Tm) 64.1 ( 0.5 °C) and trp 3 (MMP)
probe-target (Tm) 70.1 ( 1.1 °C) complexes after dissociation
revealed a difference of 6 °C in the Tm. The lowest Tmvalue was
used to determine the thermal stringency for the assay. A
temperature 14 °C below that lowest melting temperature (40 °C)
was chosen to ensure that no probe-target complexes would
dehybridize under stringent conditions.
(39) Hill, H. D.; Mirkin, C. A. Nat. Protoc. 2006, 1, 324-336.
Table 1. Oligonucleotide Sequencesa
5′-AGA CTC TAA TGC AGT CAC CAA CGC-3′
5′-TGC TCC CAA TAT AAC GTA TGC TGC-3′
5′-HS- (CH2)6-iSp18-CCG CAA TGA GTT CAA TTC ATC CGT GTA CCC-3′
5′-AAG CCA TGA GGT GAC GTA TAT TTC TTT AGT-iSp9-AGC
TAC GAA TAA-(CH2)3-SH-3′
5′-HS- (CH2)6-AAA AAA AAA ATT ATT CGT AGC T-3′
5′-ACT AAA GAA ATA TAC GTC ACC TCA TGG CTT-(iSp18)2-NH2-3′
5′-TTG AAC AAG CCG AGG GGT TCG TCT ACT GTG TAT CT-3′
5′-ATT GAC GGT CTG CTT GTT CCG GAT CTG CCA TTA GA-3′
5′-TGT TCC GGT TGC TGT AGG GTT CGG TAT ATC AAA CC-3′
5′-HS-(CH2)6-A10-AC TAA AGA AAT ATA CGT CAC CTC ATG GCT T-3′
5′-HS-(CH2)6-A10- GG GTA CAC GGA TGA ATT GAA CTC ATT GCG G-3′
5′-FITC-AA GCC ATG AGG TGA CGT ATA TTT CTT TAG T-3′
5′-FITC-CC GCA ATG AGT TCA ATT CAT CCG TGT ACC C-3′
melting Au NP
AuNP target (trp1)
MMP target (trp 3)
aiSpX ) poly(ethylene glycol) (X units of ethylene oxide).
Analytical Chemistry, Vol. 79, No. 23, December 1, 2007
Preparation of Oligonucleotide Arrays. N-Hydroxysuccin-
imide (NHS)-activated Codelink glass microscope slides (Amer-
sham Biosciences) were used to support microarrays of amine-
terminated oligonucleotides complementary to the Au particle-
bound bar code sequences and were prepared according to the
manufacturer’s protocol. The “capture” oligonucleotides were
printed in triplicate using a GME 418 robotic pin-and-ring mi-
croarrayer (Affymetrix). The chips were allowed to couple
overnight at 70% humidity and were then passivated in 0.2% SDS
at 50 °C for 30 min to hydrolyze all unreacted NHS groups.
Genomic DNA Bio Bar Code Assay. B. subtilis cells were
isolated from culture by centrifugation, and the genomic DNA
was isolated as described above using lysozyme and proteinase
K. The genomic DNA was then cut using the restriction endo-
nuclease HpyCH4V (New England Biolabs). A restriction digestion
step was needed to prevent DNA supercoiling during the heating
and subsequent detection. A dilution series of genomic DNA was
made for testing with the bio bar code assay. Additionally, an
aliquot of the genomic DNA was quantified using qPCR as
described above. Assays were assembled in nuclease-free Eppen-
dorf tubes (Ambion Inc) containing 5 µL of genomic DNA sample,
1 µL of each blocking oligonucleotide (200 µM), and 32 µL of
assay buffer. The assays were mixed thoroughly and placed at
95 °C for 10 min to denature the genomic DNA fragments. After
10 min, the temperature was lowered to 72 °C, and 10 µL of oligo-
MMPs (20 mg/mL) was added to each tube. The reactions were
mixed well and placed at 40 °C for 2 h while mixing in an end-
over-end manner to ensure that the oligo-MMPs did not settle.
The oligo-MMPs with the target bound were then washed 3 times
with 100 µL of assay buffer to remove all unbound nucleic acids
and remaining components from the restriction digest. This step
is especially important as dithiothreitol (DTT), which is used in
the restriction buffer, can react with the oligo-AuNP probes in
the next step. To the washed oligo-MMPs-target complexes 40
µL of assay buffer and 10 µL of 500 pM freshly cleaned oligo-
AuNPs were added. The reactions were vortexed and placed at
40 °C with end-over-end mixing for 1 h. The reactions were then
washed five times using 1000 µL of assay buffer to remove all
unbound oligo-AuNPs. The supernatant was removed after the
fifth wash, and the complexes were resuspended in 50 µL of 0.5
M DTT in assay buffer. The tubes were then placed at 50 °C for
15 min, followed by 45 min at 25 °C to liberate the thiolated
oligonucleotide bar codes from the surface of the gold nanoparticle
through ligand exchange.14Following bar code release, the oligo-
MMPs were isolated using a magnet and the remaining super-
natant was transferred to a new nuclease-free Eppendorf tube.
Next, 15 µL of sample was pipetted into a slide chamber well
(Nanosphere Inc.) assembled on a chip prepared as described
above. The bar code samples on the chip were heated to 60 °C
and then allowed to hybridize to their complement for 1 h at
37 °C while shaking at 120 rpm. The chips were then washed
three times in assay buffer and reassembled with clean slide
chambers. To each well, 15 µL of universal probe solution (500
pM universal AuNP, 10% formamide in assay buffer) was added.
The probes were allowed to hybridize for 45 min at 37 °C while
shaking at 120 rpm. The gaskets were then removed, and the
slides were washed twice in 0.5 M NaNO3,0.2% Tween 20, 0.1%
SDS, washed three times in 0.5 M NaNO3, and finally quickly
dipped in cold (4 °C) 0.1 M NaNO3. The slides were spun dry,
and equal parts of silver stain solution A and B (Nanosphere Inc.)
were placed on top of the slide so that the entire surface was
covered. The silver enhancement was carried out for 3 min before
being terminated by washing with Nanopure water (18 MΩ,
Barnstead). The slide was dried and imaged using a high-
resolution Verigene ID (Nanosphere Inc.), and the spot intensity
was analyzed using GenePix Software (Molecular Devices). A
representative image of the data obtained from an assay (as
displayed in false color using GenePix Software) can be seen in
Safety Considerations. To the best of our knowledge, this
assay presents no serious hazards, though caution should be taken
to avoid skin and eye contact with the silver enhancement solution.
In addition, when the assays are used in conjunction with unknown
biological samples or known bacterial samples, all proper govern-
ment safety protocols should be followed.
RESULTS AND DISCUSSION
A typical assay was performed by digesting circular genomic
DNA isolated from B. subtilis cells with 1 unit of HpyCH4V to
yield smaller linear DNA fragments, Scheme 1. In Eppendorf
Figure 1. Probe melting analysis. (A) Melting curve for the duplex formed between an oligo-AuNP probe and its fluorophore-labeled complement
(sequences given in Table 1). (B) Melting curve for the duplex formed between the oligo-MMP probe and its fluorophore-labeled complement
(sequences given in Table 1). The fluorescence of the complementary strands is quenched when they are bound to the AuNP and is recovered
when the duplexes melt with the fluorophore strand being released into solution.
Analytical Chemistry, Vol. 79, No. 23, December 1, 2007
tubes, 5 µL of target DNA at various concentrations, 1 µL of each
blocking oligonucleotide (200 µM), and 32 µL of buffer were
mixed, and the target strands were denatured at 95 °C for 10 min.
Following denaturation, the samples were cooled to 72 °C, and
10 µL of oligo-MMPs (20 mg/mL) was added to the reaction vessel
and placed on a rotating shaker at 40 °C for 2 h to facilitate target
capture. Following target capture, the samples were thoroughly
washed to remove all contaminants, and the oligo-AuNP probes
(500 pM) were added to the assay. After hybridization for 1 h at
40 °C, the sandwich complexes were washed extensively, and the
bar codes were chemically (DTT) released for Scanometric
detection.25The slides after silver amplification were imaged with
a Verigene ID system (Nanosphere Inc.), which records the
scattered light from the silver-amplified spots and provides
quantitative information regarding the concentration of bar code
To detect genomic DNA using the bio bar code assay,
separation of the duplex targets into their single-strand forms is
critical for probe binding. However, the conditions required to
thermally denature DNA are very harsh (95 °C), and the oligo-
MMPs (iron oxide nanoparticles embedded in a polymer scaffold)
deteriorate under such conditions. Chemical denaturants are not
an option, as they prevent the oligo-MMPs from hybridizing to
the target as well. To overcome the challenge of denaturing DNA
duplexes and keeping them apart long enough to allow the oligo-
MMPs to hybridize required the implementation of blocking
oligonucleotides, a modification of a strategy that has been used
in scanning probe detection techniques.40These blocking oligo-
nucleotides (blockers) consisted of three different 35-base pair
sequences, designed to flank the particle probe binding sites. In
the assay, the blockers were used in great excess (∼1:106, target/
blocker) to prevent strand rehybridization (Figure 2A).40When
the duplex DNA is heated to 95 °C with an excess of blockers,
the duplex thermally denatures, and as the solution cools, the
kinetics of blocker binding should be faster than that of native
strand rehybridization.41This should result in open regions of the
(40) Minunni, M.; Tombelli, S.; Fonti, J.; Spiriti, M. M.; Mascini, M.; Bogani, P.;
Buiatti, M. J. Am. Chem. Soc. 2005, 127, 7966-7967.
(41) Minunni, M.; Mannelli, I.; Spiriti, M. M.; Tombelli, S.; Mascini, M. Anal.
Chim. Acta 2004, 526, 19-25.
Figure 2. Blocking oligonucleotide functionality. (A) Scheme showing how the blocking oligonucleotides are designed to prevent genomic
DNA strand rehybridization. (B) This graph shows the importance of the blocking oligonucleotides to the function of the bio bar code assay. It
is clearly seen that without blockers the signal obtained in the assay is the same as that with no target, while in the presence of blockers a large
signal is obtained indicating that the genomic DNA is available for hybridization to probes.
Figure 3. Genomic DNA detection results. (A) Representative slide from a single assay showing that 2.5 fM is distinguishable from the 0 fM
(no target) sample. The gray scale image from the Verigene ID is converted to color using GenePix 6.0 software (Molecular Devices). (B) The
data shown above are the average of 5 independent runs of the genomic DNA bio bar code assay.
Analytical Chemistry, Vol. 79, No. 23, December 1, 2007
duplex. To test this hypothesis, we ran the bio bar code assay
under various conditions to determine the effectiveness of the
blockers (Figure 2B). The leftmost sample labeled “no target” was
run with digested λ-phage DNA as a negative control and 4 µM
each of the three blocking oligonucleotides. The sample in the
center was run with 250 fM target only, and the third sample (far
right) was run with 250 fM target and 4 µM each of the three
blockers. The impact of the blockers is significant. The signals
obtained for the λ-phage DNA and the target without blocking
strands fall within each other’s standard deviations, while the
target sample that contained the blockers shows a 6-fold increase
in signal. These experiments demonstrated that the blocking
oligonucleotides are critical to the success of the bio bar code
assay’s ability to detect genomic DNA.
In order to evaluate the sensitivity and dynamic range of the
assay in the presence of blockers, a digestion mixture was diluted
into a series of solutions. The bio bar code assay is capable of
detecting bacterial genomic DNA down to the low-femtomolar
concentration range, with a limit of detection of 2.5 fM (final
concentration in the assay, 7.5 × 104copies under the stated
conditions, Figure 3). The row of spots labeled 250 fM clearly
exhibits the most intense red color, indicating the strongest signal
(Figure 3A). The spots at 25 fM target concentration show an
orange/yellow color, indicating a moderate to high signal intensity.
The 2.5 fM spots show a yellow/green intensity, which is distinct
from the blue color seen at the 0 fM (no target) row at the top of
the slide. Additionally, the quantified data (5 independent experi-
ments) presented in Figure 3B show that the signal at 2.5 fM is
greater than three standard deviations above the control signal.
The normalized assay is log linear through the femtomolar
concentration range and becomes nonlinear above 1 pM due to
saturation of the scattering signal as read by the Verigene ID.
This saturation issue can be easily solved by diluting the bar codes
prior to their detection by the Scanometric method, thereby
allowing one to span the femtomolar and higher concentration
range with this method.25
We have demonstrated the ability of the bio bar code assay to
detect bacterial genomic DNA with an LOD of 2.5 fM. The
integration of blocking oligonucleotides proved to be a critical
addition to the assay, which ultimately allowed for the detection
of genomic duplex DNA isolated from B. subtilis cells. This work
paves the way for the transition of the bio bar code assay from a
laboratory technique to one that can be deployed in the field for
the rapid and accurate detection of biological terrorism agents.
B. subtilis was chosen as a model system since it is a close family
member of the lethal bacterium Bacillus anthracis, which in its
spore form is the biological weapon anthrax.42In the future, the
bio bar code assay may be coupled with automated field-
deployable sample collection technologies to potentially produce
a system for continuous biological surveillance.
C.A.M. acknowledges DARPA, HSARPA, NCI, and the NSF
for support of the work. He is also grateful for a NIH Director’s
Pioneer Award. H.D.H. acknowledges the U.S. Department of
Homeland Security (DHS) for a Graduate Fellowship under the
DHS Scholarship and Fellowship Program.
SUPPORTING INFORMATION AVAILABLE
Additional information as noted in text. This material is
available free of charge via the Internat at http://pubs.acs.org.
Received for review July 31, 2007. Accepted August 31,
(42) Atlas, R. M. Annu. Rev. Microbiol. 2002, 56, 167-185.
Scheme 1. Genomic Bio Bar Code Assaya
aThe first step is to isolate the genomic DNA from the bacterial cells and cut it with a restriction enzyme. This cut prevents the DNA
from supercoiling during heating and gives smaller target pieces. The next step is to introduce blocking oligonucleotides designed to
flank the probe binding sites and prevent strand rehybridization after thermal denaturation. The blocking oligonucleotides are used in
excess to outcompete the native strand during hybridization. The target region is now “propped” open and accessible for probe binding.
Magnetic microparticles (oligo-MMPs) are used to capture the targets from the sample and then washed. An excess of oligonucleotide-
modified gold nanoparticle probes (oligo-AuNPs) is added to the assay solutions, which results in the sandwiching of the target with lthe
oligo-MMP. Unbound oligo-AuNPs are removed by washing. The bar codes are chemically released for Scanometric detection and
Analytical Chemistry, Vol. 79, No. 23, December 1, 2007