PROTOCOL

The bio-barcode assay for the detection of protein

and nucleic acid targets using DTT-induced ligand
exchange
Haley D Hill & Chad A Mirkin

Northwestern University Department of Chemistry and The International Institute for Nanotechnology, 2145 Sheridan Road, Evanston, Illinois 60208, USA.
Correspondence should be addressed to C.A.M. ([email protected]).

Published online 29 June; corrected online 20 July 2006; doi: 10.1038/nprot.2006.51

© 2006 Nature Publishing Group http://www.nature.com/natureprotocols

The recently developed bio-barcode assay for the detection of nucleic acid and protein targets without PCR has been shown to be
extraordinarily sensitive, showing high sensitivity for both nucleic acid and protein targets. Two types of particles are used in the
assay: (i) a magnetic microparticle with recognition elements for the target of interest; and (ii) a gold nanoparticle (Au-NP) with
a second recognition agent (which can form a sandwich around the target in conjunction with the magnetic particle) and hundreds
of thiolated single-strand oligonucleotide barcodes. After reaction with the analyte, a magnetic field is used to localize and collect
the sandwich structures, and a DTT solution at elevated temperature is used to release the barcode strands. The barcode strands can
be identified on a microarray via scanometric detection or in situ if the barcodes carry with them a detectable marker. The recent
modification to the original bio-barcode assay method, utilizing DTT, has streamlined and simplified probe preparation and greatly
enhanced the quantitative capabilities of the assay. Here we report the detailed methods for performing the ligand exchange bio-
barcode assay for both nucleic acid and protein detection. In total, reagent synthesis, probe preparation and detection require 4 d.

INTRODUCTION
The ability to detect various biomarkers comprising nucleic acids disease diagnosis, tracking therapeutic efficacy, blood and food
or proteins at exceptionally low numbers is vital to the practice supply screening applications and in tracking disease recurrence.
of diagnostic medicine, the identification of biological weapons The bio-barcode assay is a promising new amplification and
agents and basic life sciences research. These biological markers detection technique that makes use of short oligonucleotides as tar-
can indicate the onset of a neurodegenerative disease, an infection get identification strands and surrogate amplification units in both
by a virus or the environmental presence of a potentially toxic or protein and nucleic acid detection1, 2 (Fig. 1). The technique uses the
lethal pathogen. High-sensitivity detection is essential for early many advantageous properties of oligonucleotide-functionalized

Figure 1 | Bio-barcode assay for DNA and protein detection. (a) Schematic representation of protein detection using the bio-barcode assay.
(b) Schematic representation of nucleic acid detection using the bio-barcode assay. (c) Schematic representation of the scanometric detection method.
Au-NP, gold nanoparticles; MMP, magnetic microparticles.

324 | VOL.1 NO.1 | 2006 | NATURE PROTOCOLS

 PROTOCOL
Au-NPs including ease of fabrication, greater oligonucleotide bind- gold probe sequences must be complementary to either the sense or
ing capabilities, stability under a variety of conditions, catalytic abi- antisense strand of the target. It is important to check the sequences
lity and optical properties3–8. The typical assay involves two types of for potential self-complementarities and hairpin formation, which
particles. One, a magnetic microparticle, has recognition elements can hinder the bio-barcode assay. The design of barcodes to use in
for the target of interest (e.g., antibody, oligonucleotide, aptamer) conjunction with protein detection is simpler than the DNA case
covalently attached to its surface. The second is usually a Au-NP and can be any sequence that the operator desires. Typically, the
that has another recognition agent, which can form a sandwich barcode is a 15mer sequence assigned to each specific protein target
around the target in conjunction with the magnetic particle. This of interest. These sequences must be checked for self-dimerization
Au-NP also carries hundreds of thiolated single-strand oligonucle- and hairpin formation, and should not end in C or G residues.
otides attached to its surface; these are the barcodes. After the two In addition to the target-specific sequence, a universal sequence
particle types have been incubated with the target and the sandwich is included if scanometric assay readout is to be used. This universal
structures have formed, a magnetic field is used to localize and col- sequence is 5′-AGC TAC GAA TAA-3′. A PEG 9mer is used between
lect them, while unattached Au-NP probes are washed away. After the universal sequence and the probe sequence to separate the two.
© 2006 Nature Publishing Group http://www.nature.com/natureprotocols

the excess Au-NPs have been removed, a DTT solution at elevated If using the fluorescence method an oligo (dA)10 sequence is used to
temperature is used to release the barcode strands from the Au-NPs space the recognition element away from the NP surface. In either
through ligand exchange9, 10. The liberated barcode strands can be case, the universal sequence or the oligo (dA)10 is placed between
identified on a microarray via scanometric detection11 with more the thiol linkage and the recognition element–barcode sequence.
NP probes, or in situ if the barcodes carry with them a detectable For mRNA detection, Dynabeads oligo (dT)25 (Invitrogen) mag-
marker (e.g., fluorophore, chemiluminescent probe, Raman active netic microparticles (MMPs) can be used for total mRNA isolation
dye or redox-active moiety)10, 12. Under controlled conditions the so as to perform the bio-barcode assay from cell lysates. The kit can
assay has shown low-attomolar (10–18) sensitivity for a variety of be used until the mRNA has been bound to the magnetic particle,
protein targets1 and high-zeptomolar (10–19) sensitivity for nucleic before continuing with the bio-barcode assay as further described.
acid targets2 when paired with scanometric readout. Antibody selection. The selection of antibodies for use in the
Producing all of the reagents for the bio-barcode assay requires bio-barcode assay can be the difference between great and poor
~3 d. Of that time, ~14 h is active and the remainder is incubation results. When choosing antibodies, it is imperative that they bind
time. This version of the bio-barcode assay requires ~9 h to per- to different epitopes on the antigen so as to form a sandwich struc-
form for nucleic acid detection and 10 h for protein detection. It ture. It is suggested that antibodies optimized for ELISA be used,
is not optimized for speed but instead to ensure proper target cap- because these antibodies are known to react with distinct epitopes.
ture and oligonucleotide hybridization. Other versions have been Generally, monoclonal antibody is conjugated to the magnetic par-
implemented that require as little as 90 min13. In total, reagent ticle, and either a monoclonal or a polyclonal antibody is used to
synthesis, probe preparation and detection require 4 d. It should generate the Au-NP probe. In certain cases, an antibody does not
be noted that probes can be stored at 4 °C for weeks at a time, and react well with the Au-NP surface and will cause formation of par-
multiple assays can be run with them. ticle aggregates or an oily film. If this is the case, try swapping the
The steps of the procedure are for the preparation of Au-NP antibody from the magnetic particle to the gold particle, and vice
(steps 1-11), the functionalization of magnetic particles with DNA versa. New antibodies will have to be chosen if the problem persists.
(steps 12-42), the functionalization of Au-NP with DNA (steps Scanometric capture probe design. The capture probe spotted
43-60) and the Bio-barcode assay for DNA detection (steps 61- on the glass slide surface for the scanometric detection is comple-
74) with options A and B for scanometric and fluorescence signal mentary to the unique 15mer barcode sequence in protein detec-
readout respectively. The protocols for magnetic particle function- tion and is complementary to t he recognition element in nucleic
alization with antibodies, Au-NP functionalization with DNA and acid detection. An amine functionality must be attached to this
antibodies and the Bio-barcode assay for protein detection are pre- capture oligonucleotide in such a way that when the barcode binds
sented in Boxes 1, 2 and 3. to the capture strand, the universal probe ‘sticky end’ extends away
from the surface into the surrounding solution. To position the
Experimental design capture strand away from the glass surface for better binding, two
Oligonucleotide probe design. Oligonucleotide probes for nucleic (PEG 18) spacers are placed between the amino-functional group
acid detection are generated using the NCBI BLAST nucleotide and the capture sequence. These oligonucleotides should be puri-
search function with the DNA sequences for a gene of interest from fied by ion exchange HPLC.
the organism in question (http://www.ncbi.nlm.nih.gov/BLAST/). Universal probe. The design work for this probe is complete.
Additionally, PCR primer design software can be used to gener- The sequence is as follows:
ate probe sequences. Traditionally these sequences are 25–35 base 5′-thiol modifier-AAA AAA AAA ATT ATT CGT AGC T-3′
pairs in length. It is important when designing sequences for use This sequence has been tested using the BLAST nucleotide func-
as probes that the end sequences of the oligonucleotides without tion and does not show high complementarity to any other DNA
the thiol (which will extend off the particle) not contain mul- sequence listed with NCBI. The universal sequence is extremely
tiple C or G residues, because these can cause particle aggrega- useful for multiplexed detection of protein or nucleic acid targets
tion. Additionally, it is desirable that the melting temperatures of within one sample14, 15.
the probes fall within a narrow range. The oligonucleotide probes Bio-barcode assay components for protein and nucleic acid
should be unique to the target, showing few or no complementa- detection. The bio-barcode assay can be performed in many
rities with other organism genomes, and both the magnetic and different media as a result of the ability to clean the samples

NATURE PROTOCOLS | VOL.1 NO.1 | 2006 | 325

 PROTOCOL
before adding the Au-NP probes. The magnetic particles allow donkey and human serum samples. In the case of DNA detec-
for the selective isolation the target of interest from a complex tion, the nucleic acid can be isolated from essentially any source.
mixture of proteins, nucleic acids, lipids, carbohydrates and (It should be noted that current investigations are continuing
other contaminants. Thus far, protein detection has been con- to optimize the protocols for protein detection in serum and
ducted in buffers, in human cerebral spinal fluid, and in goat, genomic DNA detection.)

MATERIALS
REAGENTS • Tygon tubing
NOTE: Store all reagents as suggested by the manufacturer. • Glass pipettes or plastic spatula
• Sodium citrate tribasic dihydrate >99.0% (Sigma) (HOC(COONa)(CH2CO • Teflon tape
ONa)2•2H2O) • 1,000-ml all-glass filter holder apparatus
• Hydrogen tetrachloroaurate (III) trihydrate >99.9% (Sigma) • 500-ml amber-colored glass storage container
© 2006 Nature Publishing Group http://www.nature.com/natureprotocols

(HAuCl4•3H2O) • Transmission electron microscope (TEM) may also be required

• Aqua regia cleaning solution (HCl–HNO3 (3:1)) ! CAUTION Corrosive • Magnetic separators: 2 ml, 15 ml and 50 ml
•NANOpure water (Barnstead) or other purified water (e.g., Milli-Q • Lyophilizer
(Millipore) purified)
• Orbital, rocking or rotating shaker
• Sodium phosphate monobasic, molecular biology grade (NaH2PO4)
• 15- and 50-ml conical tubes
• Sodium phosphate dibasic, molecular biology grade (Na2HPO4)
• 1.5-ml microcentrifuge tubes
• Sodium chloride, molecular biology grade (NaCl)
• 10-ml syringes
• Hydrochloric acid (HCl) ! CAUTION Can cause burns.
• 18-gauge needles
• Sodium hydroxide (NaOH) ! CAUTION Can cause burns.
• Aluminum foil
• Dynabeads M-270 Amine (Invitrogen)
• 20-ml clear borosilicate glass vials (conventionally known as EPA vials)
• Synthetic oligonucleotide probe for MMP with disulfide linker (HPLC
• pH paper and pH meter
purified)
• Eppendorf thermal mixer or temperature-controlled orbital shaker
•Anhydrous DMSO <99.9% ((CH3)2SO)
• VerigeneID (Nanosphere Inc) or traditional flatbed scanner
• DTT, molecular biology grade (C4H10O2S2)
• Hybridization gaskets, 4-well or 10-well layout (Nanosphere Inc)
• Nap-5 column (GE Healthcare)
• Slide spinner for drying
• 50 mg Succinimidyl-4-(p-maleimidophenyl)-butyrate (SMPB; Pierce)
(C18H16N2O6) • Controlled-humidity chamber
• 100 mg Sulfosuccinimidyl acetate (Sulfo-NHS-acetate; Pierce) • Fluorescence plate reader or fluorometer
(C6H6O7NSNa) • Fluorescence cuvette or 96-well Black/Clear bottom microtiter plate (Costar)
• Dynabeads M-270 Tosyl (Invitrogen) • GenePix Pro (Molecular Devices), ImageQuant (GE Healthcare) or any other
• Monoclonal antibody intensity quantification software, although GenePix is preferable
• Boric acid, molecular biology grade (H3BO3) REAGENT SETUP
• Tris, molecular biology grade (NH2C(CH2OH)3) Passivation buffer 1 150 mM phosphate buffer + 150 mM NaCl (pH = 8.0):
10.119 g Na2HPO4, 0.449 g NaH2PO4, 4.383 g NaCl, 500 ml NANOpure water.
• Ethanol (C2H5OH)
Coupling buffer 100 mM phosphate buffer + 200 mM NaCl (pH = 6.9–7.0):
• 13-nm Au colloid (synthesized in Steps 1–11 below) 4.392 g Na2HPO4, 2.291 g NaH2PO4, 5.884 g NaCl, 500 mL NANOpure water.
• Synthetic oligonucleotide probe with disulfide linker (HPLC purified) Disulfide cleavage buffer 170 mM phosphate buffer (pH = 8.0): 11.468 g
• SDS, Molecular biology grade (C12H25OSO3Na) Na2HPO4, 0.509 g NaH2PO4, 500 ml NANOpure water.
• 30-nm Au-NPs (Ted Pella, Inc.) Storage buffer 10 mM phosphate buffer + 200 mM NaCl (pH = 7.4): 0.570 g
• Polyclonal antibody or second monoclonal antibody Na2HPO4, 0.118 g NaH2PO4, 5.844 g NaCl, 500 ml NANOpure water.
• Synthetic oligonucleotide probe for Au-NP with disulfide linker (HPLC pH buffers 5 M NaOH and 5 M HCl prepared in NANOpure water.
purified) ! CAUTION Can cause burns.
Borate buffer 100 mM borate buffer (pH = 9.5): 3.091 g H3BO3, 500 ml
• Formamide, molecular biology grade (aliquot, store 4 °C) (CH3NO)
NANOpure water.
• tRNA, 10,000 units (Sigma) Washing buffer 10 mM phosphate buffer + 150 mM NaCl (pH = 7.4): 0.562 g
• Tween 20 or Polysorbate 20, molecular biology grade (C58H114O26) Na2HPO4, 0.125 g NaH2PO4, 4.383 g NaCl, 500 ml NANOpure water.
• BSA (bovine serum albumin; Ambion) Passivation buffer 2 200 mM Tris (pH = 8.5): 1.210 g Tris, 50 ml NANOpure
• Microarray slides spotted with capture oligonucleotide (4/10-well layout) water.
• Silver enhancement solutions A/B (Nanosphere Inc. or Sigma) Salting buffer 10 mM phosphate buffer + 2 M NaCl (pH 7.0): 0.0562 g
• Sodium nitrate, molecular biology grade (NaNO3) Na2HPO4, 0.0125 g NaH2PO4, 5.844 g NaCl, 50 ml NANOpure water.
Phosphate adjustment buffer 100 mM phosphate buffer (pH 7.0): 0.562 g
• Fluorophore reference standards
Na2HPO4, 0.125 g NaH2PO4, 50 ml NANOpure water.
• Fluorophore-labeled Au-NP oligonucleotide barcode Surfactant solution 10% SDS (wt/vol): 10 g SDS, 90 ml NANOpure water.
EQUIPMENT Bio-barcode assay components for protein and nucleic acid detection: There
• 0.45-µm acetate filter (Millipore) are many commercially available kits (e.g., Promega, Qiagen, Sigma) for the
• UV-visible spectrophotometer and cuvette isolation of total DNA from soil, water, feces, tissue and blood, among other
• Drying oven materials. As with any biological detection assay, sample preparation and
• 500-ml volumetric flask handling are critical. All samples should be stored at 4 °C or lower if possible to
prevent degradation from nucleases or proteases.
• 50-ml volumetric flask
Assay buffer 10 mM phosphate + 150 mM NaCl + 0.1% SDS (wt/vol)
• 1,000-ml two-neck round-bottom flask (pH = 7.4): 0.562 g Na2HPO4, 0.125 g NaH2PO4, 4.383 g NaCl, 500 mL
• Reflux condenser NANOpure water. For protein detection, omit SDS and include 0.1% BSA (wt/
• Rubber septa vol) and 0.025% Tween 20 (vol/vol).

326 | VOL.1 NO.1 | 2006 | NATURE PROTOCOLS

 PROTOCOL
Slide washing buffer A 0.5 M NaNO3 + 0.01% SDS + 0.02% Tween 20: 21.25 g EQUIPMENT SETUP
NaNO3, 500 µl 10% SDS , 100 µl Tween 20, 500 ml NANOpure water. Hybridization chamber for scanometric detection A hybridization chamber
Slide washing buffer B 0.5 M NaNO3: 21.25 g NaNO3, 500 ml NANOpure is required during the heated steps of the scanometric detection to prevent
water. evaporation from the sample wells. The chamber can be easily made. Simply
Slide washing buffer C 0.1 M NaNO3 (store at 4 °C): 2.125 g NaNO3, 500 ml place 15 ml of the assay buffer into the bottom of an empty box used to hold
NANOpure water. pipette tips, set the slides on top of the empty tip rack and place the lid on top.

PROCEDURE
13-nm Au-NP synthesis. This procedure is adapted from ref. 16
1| Clean all glassware with aqua regia, rinse copiously with NANOpure water and dry in oven at 100 °C. Wash a large stir bar
in the two-neck round-bottom flask.
! CAUTION Aqua regia and oven can cause burns.
© 2006 Nature Publishing Group http://www.nature.com/natureprotocols

2| Blow out glassware with N2 or air, and assemble reflux apparatus with the two-neck round bottom flask, reflux condenser,
Tygon tubing and rubber septa. Use Teflon tape, not grease, to protect joints. Be sure to clamp the round-bottom flask and
reflux condenser separately.

3| Prepare 500 ml of 1 mM hydrogen tetrochloroaurate (III) trihydrate with NANOpure water in the 500-ml volumetric flask
(0.1969 g Au + 500 ml H2O).
▲ CRITICAL STEP Do not use a metal spatula; use a glass pipette or plastic spatula. Metal spatulas will cause the reduction of
the gold salt onto their surface.

4| Pour the gold solution into the round-bottom flask and bring to a vigorous boil while stirring. Make sure that the water is
flowing through the reflux condenser at this time.

5| While the gold solution is heating, prepare 50 ml of 38.8 mM sodium citrate tribasic dihydrate with NANOpure water in the
clean 50-ml volumetric flask (0.5704 g sodium citrate + 50 ml H2O).

6| Once the gold solution is refluxing vigorously (1 drip s–1), remove the rubber septa and quickly add all the sodium citrate
solution. Reseal. Reflux 15 min. The solution will turn from yellow to clear, to black, to purple to deep red.

7| After 15 min, turn heat off and allow the reaction to cool to room temperature. This generally requires between 2 and 4 h.
■ PAUSE POINT Cooling overnight is acceptable; if cooling overnight, turn water flow through contender to extremely low or off.

8| Assemble 1,000-ml all-glass filter holder apparatus with the 0.45-µm acetate filter.

9| Filter cool Au-NP mixture and transfer Au-NPs into the clean amber storage bottle.

10| Measure λmax for the particles. First, blank the UV-visible spectrophotometer with NANOpure water. Then dilute 500 µl of
Au-NPs into 1 ml of NANOpure water, and take the absorbance spectrum for this sample. Well-formed 13-nm particles should
have a λmax of ~519 nm and a peak width of ~50 nm. For additional characterization, TEM is recommended.

11| Store 13-nm Au-NPs at room temperature.

Magnetic particle functionalization with DNA

12| Remove amine-functionalized MMPs from refrigerator ~15 min before use, and allow them to warm to room temperature.
Resuspend by slow vortex to avoid foaming. (Note: for antibodies, perform the steps shown in Box 1 in place of Steps 12–42.)

13| Lyophilize 25 nmol of DNA to be coupled to the MMPs.

14| Place 1 ml of the MMPs into a 1.5-ml microcentrifuge tube. Use a magnetic separator to extract MMPs to the side of the
microcentrifuge tube so as to remove supernatant. (1 ml = 30 mg MMPs, 1.75 × 10–6 mol NH2 per 1,000 mg MMPs; therefore,
5.25 × 10–6 mol NH2 per 30 mg MMP.)

15| Wash the MMPs three times with 1.5 ml DMSO using a syringe. Be sure to remove all supernatant, because the next reaction
is water sensitive.

NATURE PROTOCOLS | VOL.1 NO.1 | 2006 | 327

 PROTOCOL
16| Turn off overhead lights for Steps 17–21. The cross-linking reagent is not photostable.

17| Dissolve 50 mg SMPB in 1 ml DMSO; do this in the SMPB bottle to prevent reagent loss.

18| Add the 1 ml of SMPB–DMSO solution to dry the MMPs and resuspend them. Transfer the MMP–SMPB solution into a 50-ml
conical tube.

19| Wash the SMPB bottle twice with 2 ml DMSO and a 1.5-ml microcentrifuge tube twice with 1 ml DMSO. Add all DMSO washes
to the 50-ml conical tube containing the MMPs.

20| Add an additional 7.8 ml DMSO to the 50-ml conical tube to bring the final SMPB concentration to ~9.9 mM (15.8 ml total
volume).
© 2006 Nature Publishing Group http://www.nature.com/natureprotocols

21| Wrap the conical tube in foil and place on an orbital shaker with the tube lying on its side or rotating shaker for 4–4.5 h.
Tube must be on its side for optimal mixing.
▲ CRITICAL STEP Do not exceed 5 h. Doing so can greatly reduce coupling efficiency during the next step. Perform Steps 22–28
during this incubation.

22| Prepare 1 ml of 0.1 M DTT solution in the disulfide cleavage buffer.

▲ CRITICAL STEP This solution must be made fresh every time.

23| Add 100 µl DTT solution (Step 22) to the 25 nmol of lyophilized DNA, wrap in foil and let stand at room temperature for
2–3 h. Vortex occasionally.

24| 15 min before the completion of disulfide cleavage, begin flushing the Nap-5 column with NANOpure water. At least three
column volumes of NANOpure water must flush through before adding DNA.

25| Once all the water has run through the column, add the 100 µl of DNA–DTT to the column.

26| Allow the 100 µl to flow into the column before adding 400 µl of NANOpure water. Allow this volume to flow through the
column uncollected.

27| Add 950 µl NANOpure water to the column, and collect the flowthrough 3–4 drops at a time in 1.5-ml microcentrifuge
tubes.

BOX 1 MAGNETIC PARTICLE FUNCTIONALIZATION WITH ANTIBODIES

(i) Remove tosyl-functionalized MMPs from the refrigerator ~15 min before use. Resuspend with a slow vortex to avoid foaming.
(ii) Transfer 100 µl MMPs into a 1.5-ml microcentrifuge tube, place on magnetic rack and remove supernatant. (3 µg antibody per
107 MMPs, 2 × 109 MMPs/ ml, 100 µl = 2 × 108 MMPs; therefore, 60 µg antibody for coupling.)
(iii) Resuspend the MMPs in 1 ml borate buffer, and mix by slow tilt for 2–3 min. Repeat once.
(iv) Extract MMPs from buffer, and resuspend MMPs in 200 µl borate buffer containing 60 µg of antibodies.
(v) Incubate at 37 °C or lower depending on antibody stability for 24 h with enough mixing to keep the MMPs from settling, but
not harsh enough to denature the antibody.
(vi) Place the MMPs on the magnet and remove supernatant; save the supernatant.
(vii) Wash the MMPs twice in the washing buffer for 5 min at 4 °C.
(viii) Passivate the MMPs in 1 ml passivation buffer 2 for 24 h at 20 °C or 4 h at 37 °C.
■ PAUSE POINT This step can take place overnight.
(ix) Wash the MMPs in the washing buffer for 5 min at 4 °C. Transfer to a new tube.
(x) Store the MMPs in 1 ml of the washing buffer at 4 °C; do not freeze.
(xi) Calculate the coupling efficiency by measuring change in absorbance at 280 nm before and after the reaction; be sure to adjust
for the changes in volume. Coupling efficiency % = {[(A280 before) – (A280 after)]/(A280 before)} × 100.

328 | VOL.1 NO.1 | 2006 | NATURE PROTOCOLS

 PROTOCOL
28| Use a UV-visible spectrophotometer and the absorbance (A) at 260 nm to determine the DNA location and concentration
using Beer’s Law. Generate at least 325 µl of a 10 µM solution using the coupling buffer; it may be necessary to combine several
tubes of DNA. A = εCι, where ε is molar absorptivity, C is concentration and ι is cell path length

29| Wash the MMPs three times with 15 ml DMSO; change tubes between second and third wash. Cover particles with foil while
separating.

30| Wash the MMPs twice with coupling buffer; change tube after second wash. Cover particles with foil while separating.

31| Resuspend MMPs in 1 ml coupling buffer; take all MMPs, and place in 1.5-ml microcentrifuge tube. Use magnet to remove
supernatant.

32| Add 300 µl of the 10 µM DNA solution from Step 28 to the MMPs, close and wrap in foil. Save the remaining DNA to take a
© 2006 Nature Publishing Group http://www.nature.com/natureprotocols

precoupling concentration.

33| Shake mixture overnight at room temperature.

■ PAUSE POINT This step can be left overnight.

34| Remove supernatant and save for postcoupling efficiency calculation.

35| Transfer MMPs in 1 ml coupling buffer into a 15-ml conical tube. Wash three times with 10 mL coupling buffer. Change tube
between second and third wash.

36| Wash the MMPs twice with passivation buffer 1, changing to a 50-ml conical tube between first and second washes.

37| Dissolve 100 mg sulfo-NHS-acetate in 35 ml of passivation buffer 1. Add this solution to the dry MMPs.

38| Wrap in foil and shake on side or end over end for 1 h at room temperature.

39| Wash the MMPs three times with 20 ml passivation buffer 1, changing tubes between second and third washes.

40| Wash twice with 20 ml storage buffer, with the final resuspension in 3 ml storage buffer to give a final concentration of
10 mg ml–1.

41| Store MMPs at 4 °C. Do not freeze.

42| Calculate the coupling efficiency by measuring the change in DNA concentration before and after the reaction. Use Beer’s
Law (Step 28) and the equation: Coupling efficiency % = {[(M before) – (M after)]/(M before)} × 100, where M is molarity.

Au-NP functionalization with DNA

43| Lyophilize 5 nmol thiolated oligonucleotide probe. (Note: for DNA and antibodies, perform the steps shown in Box 2 and
Figure 2 in place of Steps 43–60.)

44| Prepare 1 ml of 0.1 M DTT solution in the disulfide cleavage buffer.

▲ CRITICAL STEP This solution must be made fresh every time.

45| Add 100 µl DTT solution (as prepared in Step 22) to the 5 nmol of lyophilized DNA, wrap in foil and let stand at room
temperature for 2–3 h. Vortex occasionally.

46| 15 min before the completion of disulfide cleavage, begin flushing a Nap-5 column with NANOpure water. At least three
column volumes of NANOpure water must flush through before adding DNA.

47| Add the 100 µl of DNA to the column after all the water has run through.

48| Once the 100 µl of DNA has flowed into the column, add 400 µl of NANOpure water to the column and allow it to flow
through uncollected.

NATURE PROTOCOLS | VOL.1 NO.1 | 2006 | 329

 PROTOCOL

BOX 2 AU-NP FUNCTIONALIZATION WITH DNA AND ANTIBODIES

(i) Lyophilize 5 nmol thiolated oligonucleotide probe.
(ii) Prepare 1 ml of 0.1 M DTT in disulfide cleavage buffer.
▲ CRITICAL STEP This solution must be made fresh
every time.
(iii) Add 100 µl DTT solution (Step ii) to the 5 nmol of
lyophilized DNA, wrap in foil and let stand at room
temperature for 2–3 h; vortex occasionally.
(iv) Pipette 10 ml 30-nm Au-NPs into a 15-ml conical vial.
(v) Adjust pH to ~9.2, and fill seven microcentrifuge tubes
(1.5 ml) with 1 ml particles each.
© 2006 Nature Publishing Group http://www.nature.com/natureprotocols

Figure 2 | Testing antibody loading. The Au-NP–antibody loading test

(vi) Add one of the following amounts of antibody to each
using salt to determine reasonable antibody amounts for probe generation.
of the microcentrifuge tubes of Au-NPs: 0 µg, 0.5 µg, From these data, 6 µg ml–1 would be an appropriate amount of antibody to
0.75 µg, 1.0 µg, 1.5 µg, 2 µg, 3 µg. Note: It may be add, because the particles to the left have aggregated, and the ones to the
necessary to test antibody amounts higher than this right have not. The lack of aggregation indicates there is too much surface
depending on the antibody, but this is a good starting coverage to leave space for the thiolated oligonucleotide barcodes to attach.
Image courtesy of Dr. A.-H. Bae.
range.
(vii) Gently vortex and incubate at room temperature for 30 min.
(viii) Add 100 µl 2 M NaCl to each tube, and watch for the red-shift indicating aggregation. Line up tubes, and select the one in which
the particles aggregate, but not immediately. This is a rough indicator for how much of the NP surface is available for thiolated
oligonucleotide binding in upcoming steps (see Fig. 2).
(ix) Rinse a 20-ml EPA vial with ethanol and then NANOpure water, and blow dry.
(x) Place 1 ml of the 30-nm NPs remaining from Step (v) at pH 9.2 into the EPA vial, and add the amount of antibody as determined
above. Vortex gently and incubate 30 min in foil.
(xi) 15 min before the end of disulfide cleavage, begin flushing a Nap-5 column with NANOpure water. At least three column volumes
of NANOpure water must flush through before adding DNA.
(xii) Add the 100 µl of DNA (Step iii) to the column after all the water has run through.
(xiii) After the 100 µl DNA has flowed into the column, add 400 µl of NANOpure water to the column and allow it to flow through
uncollected.
(xiv) Add 950 µl NANOpure water to the column, and collect the flowthrough 3–4 drops at a time in 1.5-ml microcentrifuge tubes.
(xv) Use a UV-visible spectrophotometer and the absorbance at 260 nm to determine the DNA location and concentration using Beer’s
Law: A = εCι, where ε is molar absorptivity, C is concentration and ι is cell path length.
(xvi) Add 4 nmol of the freshly cleaved thiolated oligonucleotide to the Au-NP–antibody complex. Rewrap in foil and incubate 4 min at
10 °C.
(xvii) Add phosphate adjustment buffer to the NP solution to obtain a final phosphate concentration of 9 mM. Calculation: 1,000 µl
Au-NPs + x µl DNA = total volume in microliters. (Total volume in µl)/10 = y µl phosphate adjustment buffer needed.
(xviii) Rewrap in foil and place on orbital shaker for 30 min.
(xix) Calculate amount of salting buffer needed to obtain a final concentration of 0.15 M NaCl. Calculation: (Total volume 2 × 0.3 M)/(2 M)
= volume of salting buffer needed in microliters. No. of additions = 6 amount per addition = salt/6. The number of additions of salting
buffer is equal to 6, therefore the amount per addition is equal to the volume of salting buffer divided by 6.
(xx) Over the course of 3 h, make six additions of one-sixth of the total salting buffer needed to reach a final concentration of 0.15 M
NaCl. Do the additions while shaking gently, alternatively, on a low vortex speed.
(xxi) Incubate 1 h.
(xxii) Centrifuge the gold particles for 15 min at 10,000g at 4 °C; remove supernatant. Resuspend in 1 ml of washing buffer. Repeat Step
(xxii) twice.
(xxiii) Centrifuge particles for 15 min at 10,000g at 4 °C; remove supernatant. Resuspend in 200 µl of washing buffer and store at 4 °C.
Do not freeze.

49| Then add 950 µl NANOpure water to the column, and collect the flowthrough 3–4 drops at a time in 1.5-ml microcentrifuge
tubes.

50| Use a UV-visible spectrophotometer and the absorbance at 260 nm to determine the DNA location and concentration using
Beer’s Law: A = εCι, where ε is molar absorptivity, C is concentration and ι is cell path length.

330 | VOL.1 NO.1 | 2006 | NATURE PROTOCOLS

 PROTOCOL
51| Rinse a 20-ml EPA vial with ethanol and then NANOpure water; blow dry.

52| Add 1 ml 13-nm Au-NPs synthesized previously.

53| Calculate the number of moles of oligonucleotide per tube, and add 4 nmol of the freshly reduced thiolated
oligonucleotide to the Au-NPs. Record the volume.

54| Wrap in foil and place on orbital shaker overnight at room temperature.
■ PAUSE POINT This step can be left overnight.

55| Add phosphate adjustment buffer to the NP solution to obtain a final phosphate concentration of 9 mM. Calculation:
1,000 µl AuNPs + x µl DNA = total volume in µl. (Total volume 1 in µl)/10 = y µl phosphate adjustment buffer needed.
© 2006 Nature Publishing Group http://www.nature.com/natureprotocols

56| Add surfactant solution to obtain a final SDS concentration of ~0.1% (wt/vol) . This helps to keep the particles from
aggregating and adds to the efficiency of washing them in future steps. Calculation: 1000 µl AuNPs + x µl DNA + y µl
phosphate adjustment buffer = total volume 2. SDS to add = (total volume 2 × 0.1)/10.

57| Rewrap in foil and place on an orbital shaker for 30 min.

58| Calculate the volume of salting buffer needed to obtain a final concentration of 0.3 M NaCl. Calculation:
(Total volume 2 × 0.3 M)/(2M) = volume of salting buffer needed in microliters. The number of additions of salting buffer is
equal to 6, therefore the amount per addition is equal to the volume of salting buffer divided by 6.

59| Over the course of 2 d, make six additions of one-sixth of the total salting buffer needed to reach a final concentration
of 0.3 M NaCl. Do the additions while shaking gently or on a low vortex speed.

60| After the last salt addition, allow the particles to equilibrate overnight. Well-functionalized Au-NPs should be the same
color as the un-modified Au-NPs with no visible aggregates.
■ PAUSE POINT Particles can be stored at room temperature for as long as 1 month in this state.

Bio-barcode assay for DNA detection

61| Determine the number of samples to be tested, including one for the negative control (×). Add 20× µl 10 mg ml–1 MMPs
to a 1.5-ml microcentrifuge tube (see ref. 9). (Note: for protein detection, perform the steps shown in Box 3 (see ref. 13) in
place of Steps 61–74.)

62| Wash the MMPs twice with the assay buffer. Resuspend MMPs in half the original volume of assay buffer to gain a
concentration of 20 mg ml–1. Omit this step if using the oligo (dT)25 kit from Dynal, and follow the kit instructions until the
mRNA is isolated.

63| Mix in 1.5-ml microcentrifuge tubes: (i) 30 µl assay buffer; (ii) 10 µl MMP solution; (iii) 10 µl target solution.

64| Shake the reactions at a temperature ~15 °C below the melting point of the DNA for 45 min. A longer incubation period
may be needed for samples that are more complex. Shake sufficiently so that the MMPs do not settle.

65| During the incubation, centrifuge 100 µl of Au-NP probe at 13,000g for 20 min. Remove the supernatant and resuspend
in 500 µl assay buffer. Repeat Step 65 four times.

66| After the final spin/wash in Step 65, resuspend the gold particles in assay buffer to generate a 1 nM solution. Calculate
the Au-NP concentration using Beer’s Law (Step 28) and a molar absorptivity at 519 nm of 2.7 × 108 liter mol–1∙cm–1.

67| Wash the magnetic particles with target twice with the assay buffer, and resuspend in 50 µl of assay buffer. Wait 3 min
between each wash while the magnet isolates the MMPs.

68| Add 10 µl of Au-NP probes recently washed (Step 66).

69| Incubate at the same temperature as in Step 64 for 1.5–2 h with shaking so that the MMPs do not settle.

NATURE PROTOCOLS | VOL.1 NO.1 | 2006 | 331

 PROTOCOL

BOX 3 BIO-BARCODE ASSAY FOR PROTEIN DETECTION

(i) Determine the number of samples to be tested, including one for the negative control (x). Add 20x µl 10 mg ml–1 MMPs to a
1.5-ml microcentrifuge tube.
(ii) Wash MMPs twice with the assay buffer. Redisperse the MMPs in twice their original volume of assay buffer.
(iii) Mix in 1.5-mL microcentrifuge tubes: (a) 150 µl assay buffer, (b) 40 µl MMP solution, (c) 10 µl target solution.
(iv) Vortex and incubate for 1.5 h at 1,400 r.p.m. at 25 °C.
(v) Place on magnet and remove supernatant; wash twice with 200 µl assay buffer.
(vi) Prepare tRNA solution at 66 mg ml–1 in NANOpure water (store at 4 °C). Note: The tRNA is used to reduce nonspecific binding
between the antibodies on the gold and magnetic particles.
(vii) Add 50 µl 30-nm Au-NP solution prepared by mixing (a) 215 µl assay buffer, (b) 25 µl tRNA, (c) 10 µl 10 nM Au-NPs in assay
buffer.
© 2006 Nature Publishing Group http://www.nature.com/natureprotocols

(viii) Centrifuge for 1 h at 1,400 r.p.m. at 25 °C.

(ix) Add 200 µl of assay buffer.
(x) Wash seven times with 200 µl of assay buffer. Wait 3 min between each wash while the magnet isolates the MMPs.
(xi) Release barcodes in 50 µl of DTT solution prepared in the assay buffer. Note: Increasing the salt concentration in the assay
buffer for the remainder of the assay may improve results depending on barcode sequences.
(xii) Heat the tubes for 15 min at 50 °C, and then incubate for 45 min at 25 °C under vortex.
(xiii) Choose appropriate signal readout method protocol, scanometric or fluorescence.
■ PAUSE POINT The bio-barcode assay can be stopped after the release of the barcodes by DTT. To stop here, extract MMPs
from supernatant and freeze samples at –20 °C.

70| Wash the detections five times with 100 µl assay buffer. Wait 3 min between each wash while the magnet isolates
the MMPs.
▲ CRITICAL STEP Be sure to remove all unbound Au-NPs to be sure that all signal seen is due to specific target binding
events.

71| Prepare a 0.5 M DTT solution in the assay buffer. Note: This solution must be made fresh each day that the Barcode
assay is performed. Increasing the salt concentration in the assay buffer for the remainder of the assay may improve
scanometric results depending on barcode sequences. DTT is used because it is a less foul-smelling reducing agent than
2-mercaptoethanol.

72| Resuspend the MMP-target–Au-NP complexes in 50 µl DTT buffer from Step 71 and vortex.

73| Incubate samples at 50 °C for 15 min, and then 45 min at 25 °C under vortex.

74| Choose appropriate signal readout method protocol: scanometric (A) or fluorescence (B).
■ PAUSE POINT The bio-barcode assay can be stopped after the release of the barcodes by DTT. To stop here, remove MMPs
and Au-NPs from supernatant and freeze samples at –20 °C. Do not leave the barcodes in solution with the magnetic particles,
because this will increase noise when using the scanometric method.
(A) Scanometric detection of the barcodes (DNA and protein detection)
(i) During the barcode release step, passivate slides spotted for scanometric detection with a 0.2% SDS (wt/vol) solution
made in NANOpure water. Place 40 ml SDS solution in 50-ml conical tube and add slide. This removes the unreacted NHS
groups from the surface of the slides.
(ii) Incubate at 50 °C for 15 min, wash in NANOpure water and spin dry.
(iii) Assemble the chip in the holder in the hybridization chamber.
(iv) After barcode release, extract the magnetic particles on the magnet for 3 min, and transfer the supernatants to clean
microcentrifuge tubes, or remove samples from freezer and thaw.
(v) Spin the supernatants for 5 min at 13,000g to pellet the aggregated gold particles.
(vi) Add 15–20 µl of the supernatant to a freshly rehydrated, passivated slide. The volume depends on the slide
configuration. There should be one detection per well.
(vii) Incubate for 15 min at 60 °C, 30 min at 37 °C and 15 min at 25 °C on the shaker (120 r.p.m.) in a controlled-humidity
chamber.

332 | VOL.1 NO.1 | 2006 | NATURE PROTOCOLS

 PROTOCOL
(viii) During the incubation in Step vii, spin 100 µl of universal Au-NP probe at 13,000g for 20 min. Remove the supernatant
and resuspend in 500 µl assay buffer. Repeat Step viii four times.
(ix) Remove slides from shaker and gaskets and wash slides three times in 40 ml of assay buffer in 50-ml conical tubes for
2 min in each.
(x) Spin slides nearly dry, and reassemble apparatus with a new top gasket.
▲ CRITICAL STEP A new gasket is vital, because the DTT on the one used in Step vii can cause the universal probes to
aggregate.
(xi) Add 15–20 µl of the universal probe solution to each well. The 13-nm probe solution should be composed of 500 pM
13-nm Au-NPs in assay buffer plus formamide. Again follow Step 66 to determine NP probe concentration. Note: Formamide
percentage should be determined depending on the specific system. It works well to begin with 10% and work up or down
from there. Formamide is a stringency reagent that lowers the nonspecific interaction between oligonucleotides.
(xii) Fill each well with 15 µl universal probe solution from Step xi.
(xiii) Allow the probe to associate with the chip for 45 min at 37 °C in the controlled-humidity chamber with shaking at
© 2006 Nature Publishing Group http://www.nature.com/natureprotocols

120 r.p.m.
(xiv) Disassemble apparatus, and wash the slides individually twice in 40 ml of slide washing buffer A in a conical tube for 1 min.
(xv) Next, wash the slides three times in 40 ml slide washing buffer B for 1 min at a time.
(xvi) Finally, quickly immerse the slides in 40 ml cold slide washing buffer C, and spin dry.
(xvii) Lay the slide flat on the benchtop on a folded Kimwipe.
(xviii) Prepare 2 ml silver staining solution (1 ml A and 1 ml B), and mix well.
! CAUTION Be sure not to cross-contaminate the stock solutions of A and B.
(xix) Pipette silver stain onto slide, ensuring that the lower two-thirds of the slide are completely covered.
(xx) Develop for 2–4.5 min depending on the initial target concentration. Once a silver staining time has been determined,
all remaining experiments to be compared must use the same staining time. Do not silver stain so that the spots
are visible with the naked eye, because this indicates they have become saturated and can no longer be accurately
quantified.
(xxi) Wash slide thoroughly with NANOpure water, and spin dry.
(xxii) Image with Verigene-ID or conventional flatbed scanner, and save (Figs. 3 and 4).
(xxiii) Quantify spot intensity using quantification software.
(B) Fluorescence detection of the barcodes (DNA and protein detection)
(i) After barcode release, extract the magnetic particles down on the magnet for 3 min and transfer the supernatants to
clean microcentrifuge tubes.
(ii) Spin the supernatants for 5 min at 13,000g to pellet the aggregated gold particles.
(iii) Prepare serial dilution of known concentrations in barcode release buffer to generate a calibration curve.
(iv) Remove supernatant from gold pellet and place in a new microcentrifuge tube.
(v) Adjust the volume of released barcodes to 100 µl using additional barcode release buffer.
(vi) There are two methods for filling a 96-well plate: either fill wells with 100 µl of each sample and standards, skipping
every other well and filling remaining empty wells with 100 µl of NANOpure water, or use cuvette to measure each
sample individually.
(vii) Measure fluorescence and perform data analysis.

● TIMING
13-nm Au-NP synthesis
Steps 1–6, 2 h
Step 7, 3 h
Steps 8–11, 20 min
Total time ~5.5 h

MMP functionalization with DNA

Steps 13-20, 45 min
Steps 21-24, 4 h
Steps 25-32, 1.5 h
Step 33, overnight
Steps 34-37, 1 h
Step 38, 1 h
Steps 39-42, 45 min
Total time ~21 h (including overnight = 12 h)

NATURE PROTOCOLS | VOL.1 NO.1 | 2006 | 333

 PROTOCOL
Au-NP functionalization with DNA
Steps 43-53, 3 h
Step 54, Overnight (12 h)
Steps 55-56, 10 min
Step 57, 30 min
Steps 58-60, 2 d
Total time ~3 d (4 h active time,
remainder incubations)

Bio-Barcode assay for DNA detection

Steps 61-63, 20 min
Steps 64-66, 45 min
Steps 67-68, 15 min
© 2006 Nature Publishing Group http://www.nature.com/natureprotocols

Step 69, 2 h
Steps 70-72, 1.5 h
Step 73, 1 h
Total time ~5.5 h

(A) Scanometric detection of

the barcodes (DNA and protein
detection)
Steps (i)-(vi), 1 h 15 min
Steps (vii)-(viii), 1 h Figure 3 | Scanometric image. This image shows Figure 4 | DNA detection image. This image
Steps (ix)-(xii), 15 min how slides should look after silver enhancement shows how the data should look after running
Step (xiii), 45 min as seen by the VerigeneID. The image shows the bio-barcode assay and completing the silver
Steps (xiv)-(xxiii), 45 min the scattering of the silver-enhanced spots on enhancement as seen using a false coloring
Total time ~4 h the slide. Row 1, negative control; row 2, high- scheme with the GenePix software. Row 1, negative
femtomolar target; row 3, mid-femtomolar target; control; row 2, high-femtomolar target; row 3, mid-
row 4, low-femtomolar target. femtomolar target; row 4, low-femtomolar target.
(B) Fluorescence detection of
the barcodes (DNA and protein
detection)
Total time ~1 h

MMP functionalization with antibodies (Box 1)

Steps (i)-(iv), 30 min
Step (v), 24 h
Steps (vi)-(vii), 10 min
Step (viii), 4–24 h
Steps (ix)-(xi), 30 min
Total time minimum ~29 h with one 24-h incubation; can be as long as 59 h.

Au-NP functionalization with DNA and antibodies (Box 2)

Step (i), 2 h
Steps (ii)-(iii), 2 h
Steps (iv)-(xx), 2 h
Step (xxi), 1 h
Steps (xxii)-(xxiii), 1.5 h
Total time ~9 h

Bio-Barcode assay for protein detection (Box 3)

Steps (i)-(iii), 20 min
Step (iv), 1.5 h
Steps (v)-(vii), 15 min
Step (viii), 1 h
Steps (ix)-(xii), 1.5 h
Total time ~4.5 h

334 | VOL.1 NO.1 | 2006 | NATURE PROTOCOLS

 PROTOCOL
? TROUBLESHOOTING
Nonspecific binding between Au-NPs and magnetic microparticles can be addressed by using varying amounts of
formamide during the Au-NP probe hybridization to the target. It is advisable to test a range of concentrations to
determine which amount of formamide effectively eliminates nonspecific binding. Generally, between 5 and 20%
formamide will solve the problem. The formamide should be stored at 4 °C in aliquots to limit degradation. For RNA
detection, be sure to purchase RNase-free formamide. Occasionally, preparation of the antibody-functionalized gold
probes will fail because of antibody denaturation. Aliquot antibodies upon their arrival, and avoid successive freeze/
thaws. See Table 1 for other troubleshooting options.
To perform the bio-barcode assay for RNA detection, it is important that all components be RNase free. NANOpure
water should be treated with diethyl-pyrocarbonate (DEPC), autoclaved twice and stirred at room temperature for 1–2 d.
This water can then be substituted in all steps of the procedure. Use RNase-free plastic wear and reagents, and treat all
glassware to remove RNases.
The most critical steps in the bio-barcode assay are the washing steps. It is vital that none of the magnetic
© 2006 Nature Publishing Group http://www.nature.com/natureprotocols

microparticles be accidentally removed. To prevent the loss of magnetic particles and sandwich complexes, one must draw
supernatant off the particles slowly and carefully. During washing it is important not to vortex the microcentrifuge tubes
too vigorously to avoid disturbing the hybridization. Additional critical steps are noted in the text.

TABLE 1 | Troubleshooting guide for the bio-barcode assay.

PROBLEM POSSIBLE REASON SOLUTION
High negative-control signal Nonspecific binding between Try various concentrations of formamide during Au-NP hybridization and
MMPs and Au-NPs wash steps to increase stringency (5%–20%).
Try increasing the temperature during hybridization steps to increase
stringency further.
Add 0.1% bovine serum albumin to the assay buffer in order to passivate
magnetic microparticles.
Gold probes are old; remake.
Try additional washings after the Au-NP hybridization.
Antibodies are denatured and are interacting nonspecifically; remake
probe sets.
High negative-control signal Poor oligonucleotide probe MMP sequence and Au-NP sequence could be interacting; check
design sequences. Then try including formamide to reduce hydrogen bonding if
this could be a problem.
If formamide does not solve the problem, redesign one of the probes, and
be sure to check cross-complementarity.
No signal (expected) Antibodies denatured Remake antibody probes.
No signal (expected) Stringency too high for DNA Lower the temperature at which the assay is run.
binding in assay
No signal (expected) Old silver stain solution Use new silver stain.
High slide background Salt contamination Wash slides longer in wash buffers before staining.
High slide background Insufficient passivation Increase passivation time in 0.2% SDS to 45 min.
Nanoparticles aggregate Salt additions are too large Instead of 6 additions, try 10 or 15 over the same time course.
during salting
Increase the time course if smaller additions do not work.
Check that the oligonucleotide sequence does not contain a guanine
residue at the end of the strand extending away from the nanoparticle
surface. If it does, heat probes to 37 °C.
Nanoparticle aggregate Antibodies are not compatible In some cases, antibodies cause the nanoparticles to form an oily film.
during salting with electrostatic attachment Attempt to remake the probe once. Also try switching MMP and Au-NP
to Au-NP antibodies.

NATURE PROTOCOLS | VOL.1 NO.1 | 2006 | 335

 PROTOCOL
TABLE 1 | Troubleshooting guide for the bio-barcode assay (continued).
Synthesized Au-NPs Reaction not refluxing Be sure that the reflux of the gold salt is steady and rapid before the
appear purple or have wide vigorously enough addition of the citrate solution.
absorption peak
Add citrate solution as quickly, in 15 s or less.
Scanometric intensity levels Signal saturation Try diluting the barcodes before adding them to the slides. This should
plateau at higher targets not be a problem for fluorescence detection.
concentrations
Above are common problems that occur when running the bio-barcode assay, along with their general causes and solutions. It is most
useful to change only one parameter at a time until the problem is solved. Additionally, be sure that the target samples have been handled
properly and are not degraded or denatured.
© 2006 Nature Publishing Group http://www.nature.com/natureprotocols

ANTICIPATED RESULTS
Figure 3 shows results of outstanding scanometric data. Figure 4 shows excellent data from a DNA detection using the
bio-barcode assay. Additional data can be seen by consulting refs. 1, 2, 4 and 10.

ACKNOWLEDGMENTS C.A.M. acknowledges HSARPA, NIH Pioneer Award, NIH dependent stability of DNA-modified gold nanoparticles. Langmuir 18,
NIAID, AFOSR, Doris Duke Charitable Foundation, NSF/NSEC and NCI. H.D.H: This 6666–6670 (2002).
work was performed while on appointment as a U.S. Department of Homeland 6. Jin, R.C. et al. What controls the melting properties of DNA-linked gold
Security (DHS) Fellow under the DHS Scholarship and Fellowship Program, a nanoparticle assemblies? J. Am. Chem. Soc. 125, 1643–1654 (2003).
program administered by the Oak Ridge Institute for Science and Education 7. Lytton-Jean, A.K.R. & Mirkin, C.A. A thermodynamic investigation into
(ORISE) for DHS through an interagency agreement with the U.S. Department of the binding properties of DNA functionalized gold nanoparticle probes and
Energy (DOE). ORISE is managed by Oak Ridge Associated Universities under DOE molecular fluorophore probes. J. Am. Chem. Soc. 127, 12754–12755 (2005).
contract number DE-AC05-06OR23100. All opinions expressed in this paper are 8. Demers, L.M. et al. Thermal desorption behavior and binding properties of DNA
the author’s and do not necessarily reflect the policies and views of DHS, DOE or bases and nucleosides on gold. J. Am. Chem. Soc. 124, 11248-11249 (2002).
ORISE. 9. Thaxton, C.S. et al. A bio-bar-code assay based upon dithiothreitol-induced
oligonucleotide release. Anal. Chem. 77, 8174–8178 (2005).
COMPETING INTERESTS STATEMENT The authors declare that they have no 10. Demers, L.M. et al. A fluorescence-based method for determining the surface
competing financial interests. coverage and hybridization efficiency of thiol-capped oligonucleotides bound
to gold thin films and nanoparticles. Anal. Chem. 72, 5535–5541 (2000).
Published online at http://www.natureprotocols.com 11. Taton, T.A., Mirkin, C.A. & Letsinger, R.L. Scanometric DNA array detection with
Rights and permissions information is available online at http://npg.nature.com/ nanoparticle probes, Science 289, 1757–1760 (2000).
reprintsandpermissions 12. Cao, Y.W.C., Jin, R.C. & Mirkin, C.A. Nanoparticles with Raman spectroscopic
fingerprints for DNA and RNA detection. Science 297, 1536–1540 (2002).
1. Nam, J.M., Thaxton, C.S. & Mirkin, C.A. Nanoparticle-based bio-bar codes for 13. Oh, B.K., Nam, J.M., Lee, S.W. & Mirkin, C.A. A fluorophore-based bio-barcode
the ultrasensitive detection of proteins. Science 301, 1884–1886 (2003). amplification assay for proteins, Small 2, 103–108 (2006).
2. Nam, J.M., Stoeva, S.I. & Mirkin, C.A. Bio-bar code–based DNA detection 14. Stoeva, S.I. et al. Multiplexed detection of protein cancer markers with bio-
with PCR-like sensitivity, J. Am. Chem. Soc. 126, 5932–5933 (2004). barcoded nanoparticle probes, J. Am. Chem. Soc. (2006)
3. Mirkin, C.A., Letsinger, R.L., Mucic, R.C. & Storhoff, J.J. A DNA-based method DI: 10.1020/ja0613106.
for rationally assembling nanoparticles into macroscopic materials, Nature 15. Stoeva, S.I., Lee, J.-S., Thaxton, C.S. & Mirkin, C.A. Multiplexed DNA detection
382, 607–609 (1996). with biobarcoded nanoparticle probes. Angew. Chem. Int. Ed. Engl. 45,
4. Storhoff, J.J. et al. What controls the optical properties of DNA-linked gold 3303–3306 (2006).
nanoparticle assemblies? J. Am. Chem. Soc. 122, 4640–4650 (2000). 16. Frens, G. Controlled nucleation for regulation of particle-size in monodisperse
5. Storhofff, J.J., Elghanian, R., Mirkin, C.A. & Letsinger, R.L. Sequence- gold suspensions. Nat. Phys. Sci. 241, 20–22 (1973).

336 | VOL.1 NO.1 | 2006 | NATURE PROTOCOLS

 ERRATUM

Erratum: The bio-barcode assay for the detection of

protein and nucleic acid targets using DTT-induced
ligand exchange
Haley D Hill & Chad A Mirkin
Nat. Protocols 1, 324–336 (2006); published online 29 June; corrected online 20 July 2006.

In the version of the article initially published online, incorrect (non-final) versions of the article were posted in both the PDF
and HTML formats. The errors have been corrected in all versions of the article.
© 2006 Nature Publishing Group http://www.nature.com/natureprotocols