Protocol


Nature Protocols 1, 324 - 336 (2006)
Published online: 29 June 2006 | Corrected online: 1 August 0608 | doi:10.1038/nprot.2006.51

Subject Categories: Biochemistry and protein analysis | Nanotechnology | Synthetic chemistry

The bio-barcode assay for the detection of protein and nucleic acid targets using DTT-induced ligand exchange

Haley D Hill1 & Chad A Mirkin1

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.

*Note: 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.

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Introduction

The ability to detect various biomarkers comprising nucleic acids or proteins at exceptionally low numbers is vital to the practice of diagnostic medicine, the identification of biological weapons agents and basic life sciences research. These biological markers can indicate the onset of a neurodegenerative disease, an infection by a virus or the environmental presence of a potentially toxic or lethal pathogen. High-sensitivity detection is essential for early disease diagnosis, tracking therapeutic efficacy, blood and food supply screening applications and in tracking disease recurrence.

The bio-barcode assay is a promising new amplification and detection technique that makes use of short oligonucleotides as target identification strands and surrogate amplification units in both protein and nucleic acid detection1, 2 (Fig. 1). The technique uses the many advantageous properties of oligonucleotide-functionalized Au-NPs including ease of fabrication, greater oligonucleotide binding capabilities, stability under a variety of conditions, catalytic ability and optical properties3, 4, 5, 6, 7, 8. The typical assay involves two types of particles. One, a magnetic microparticle, has recognition elements for the target of interest (e.g., antibody, oligonucleotide, aptamer) covalently attached to its surface. The second is usually a Au-NP that has another recognition agent, which can form a sandwich around the target in conjunction with the magnetic particle. This Au-NP also carries hundreds of thiolated single-strand oligonucleotides attached to its surface; these are the barcodes. After the two particle types have been incubated with the target and the sandwich structures have formed, a magnetic field is used to localize and collect them, while unattached Au-NP probes are washed away. After the excess Au-NPs have been removed, a DTT solution at elevated temperature is used to release the barcode strands from the Au-NPs through ligand exchange9, 10. The liberated barcode strands can be identified on a microarray via scanometric detection11 with more NP probes, or in situ if the barcodes carry with them a detectable marker (e.g., fluorophore, chemiluminescent probe, Raman active dye or redox-active moiety)10, 12. Under controlled conditions the assay has shown low-attomolar (10−18) sensitivity for a variety of protein targets1 and high-zeptomolar (10−19) sensitivity for nucleic acid targets2 when paired with scanometric readout.

Figure 1: Bio-barcode assay for DNA and protein detection.
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.

Full size image (173 KB)

Producing all of the reagents for the bio-barcode assay requires ~3 d. Of that time, ~14 h is active and the remainder is incubation time. This version of the bio-barcode assay requires ~9 h to perform for nucleic acid detection and 10 h for protein detection. It is not optimized for speed but instead to ensure proper target capture and oligonucleotide hybridization. Other versions have been implemented that require as little as 90 min13. In total, reagent synthesis, probe preparation and detection require 4 d. It should be noted that probes can be stored at 4 °C for weeks at a time, and multiple assays can be run with them.

The steps of the procedure are for the preparation of Au-NP (steps 1-11), the functionalization of magnetic particles with DNA (steps 12-42), the functionalization of Au-NP with DNA (steps 43-60) and the Bio-barcode assay for DNA detection (steps 61-74) with options A and B for scanometric and fluorescence signal readout respectively. The protocols for magnetic particle functionalization with antibodies, Au-NP functionalization with DNA and antibodies and the Bio-barcode assay for protein detection are presented in Boxes 1, 2 and 3.

Experimental design

Oligonucleotide probe design. Oligonucleotide probes for nucleic acid detection are generated using the NCBI BLAST nucleotide search function with the DNA sequences for a gene of interest from the organism in question (http://www.ncbi.nlm.nih.gov/BLAST/). Additionally, PCR primer design software can be used to generate probe sequences. Traditionally these sequences are 25–35 base pairs in length. It is important when designing sequences for use as probes that the end sequences of the oligonucleotides without the thiol (which will extend off the particle) not contain multiple C or G residues, because these can cause particle aggregation. Additionally, it is desirable that the melting temperatures of the probes fall within a narrow range. The oligonucleotide probes should be unique to the target, showing few or no complementarities with other organism genomes, and both the magnetic and gold probe sequences must be complementary to either the sense or antisense strand of the target. It is important to check the sequences for potential self-complementarities and hairpin formation, which can hinder the bio-barcode assay. The design of barcodes to use in conjunction with protein detection is simpler than the DNA case and can be any sequence that the operator desires. Typically, the barcode is a 15mer sequence assigned to each specific protein target of interest. These sequences must be checked for self-dimerization and hairpin formation, and should not end in C or G residues.

In addition to the target-specific sequence, a universal sequence is included if scanometric assay readout is to be used. This universal sequence is 5′-AGC TAC GAA TAA-3′. A PEG 9mer is used between the universal sequence and the probe sequence to separate the two. If using the fluorescence method an oligo (dA)10 sequence is used to space the recognition element away from the NP surface. In either case, the universal sequence or the oligo (dA)10 is placed between the thiol linkage and the recognition element–barcode sequence.

For mRNA detection, Dynabeads oligo (dT)25 (Invitrogen) magnetic microparticles (MMPs) can be used for total mRNA isolation so as to perform the bio-barcode assay from cell lysates. The kit can be used until the mRNA has been bound to the magnetic particle, before continuing with the bio-barcode assay as further described.

Antibody selection. The selection of antibodies for use in the bio-barcode assay can be the difference between great and poor results. When choosing antibodies, it is imperative that they bind to different epitopes on the antigen so as to form a sandwich structure. It is suggested that antibodies optimized for ELISA be used, because these antibodies are known to react with distinct epitopes. Generally, monoclonal antibody is conjugated to the magnetic particle, and either a monoclonal or a polyclonal antibody is used to generate the Au-NP probe. In certain cases, an antibody does not react well with the Au-NP surface and will cause formation of particle aggregates or an oily film. If this is the case, try swapping the antibody from the magnetic particle to the gold particle, and vice versa. New antibodies will have to be chosen if the problem persists.

Scanometric capture probe design. The capture probe spotted on the glass slide surface for the scanometric detection is complementary to the unique 15mer barcode sequence in protein detection and is complementary to the recognition element in nucleic acid detection. An amine functionality must be attached to this capture oligonucleotide in such a way that when the barcode binds to the capture strand, the universal probe 'sticky end' extends away from the surface into the surrounding solution. To position the capture strand away from the glass surface for better binding, two (PEG 18) spacers are placed between the amino-functional group and the capture sequence. These oligonucleotides should be purified by ion exchange HPLC.

Universal probe. The design work for this probe is complete. The sequence is as follows:

5′-thiol modifier-AAA AAA AAA ATT ATT CGT AGC T-3′

This sequence has been tested using the BLAST nucleotide function and does not show high complementarity to any other DNA sequence listed with NCBI. The universal sequence is extremely useful for multiplexed detection of protein or nucleic acid targets within one sample14, 15.

Bio-barcode assay components for protein and nucleic acid detection. The bio-barcode assay can be performed in many different media as a result of the ability to clean the samples before adding the Au-NP probes. The magnetic particles allow for the selective isolation the target of interest from a complex mixture of proteins, nucleic acids, lipids, carbohydrates and other contaminants. Thus far, protein detection has been conducted in buffers, in human cerebral spinal fluid, and in goat, donkey and human serum samples. In the case of DNA detection, the nucleic acid can be isolated from essentially any source. (It should be noted that current investigations are continuing to optimize the protocols for protein detection in serum and genomic DNA detection.)


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Materials

Reagents

  • NOTE: Store all reagents as suggested by the manufacturer.
  • Sodium citrate tribasic dihydrate >99.0% (Sigma) (HOC(COONa)(CH2COONa)2•2H2O)
  • Hydrogen tetrachloroaurate (III) trihydrate >99.9% (Sigma) (HAuCl4•3H2O)
  • Aqua regia cleaning solution (HCl–HNO3 (3:1))
    Caution Corrosive
  • NANOpure water (Barnstead) or other purified water (e.g., Milli-Q (Millipore) purified)
  • Sodium phosphate monobasic, molecular biology grade (NaH2PO4)
  • Sodium phosphate dibasic, molecular biology grade (Na2HPO4)
  • Sodium chloride, molecular biology grade (NaCl)
  • Hydrochloric acid (HCl)
    Caution Can cause burns.
  • Sodium hydroxide (NaOH)
    Caution Can cause burns.
  • Dynabeads M-270 Amine (Invitrogen)
  • Synthetic oligonucleotide probe for MMP with disulfide linker (HPLC purified)
  • Anhydrous DMSO <99.9% ((CH3)2SO)
  • DTT, molecular biology grade (C4H10O2S2)
  • Nap-5 column (GE Healthcare)
  • 50 mg Succinimidyl-4-(p-maleimidophenyl)-butyrate (SMPB; Pierce) (C18H16N2O6)
  • 100 mg Sulfosuccinimidyl acetate ( Sulfo-NHS-acetate; Pierce) (C6H6O7NSNa)
  • Dynabeads M-270 Tosyl (Invitrogen)
  • Monoclonal antibody
  • Boric acid, molecular biology grade (H3BO3)
  • Tris, molecular biology grade (NH2C(CH2OH)3)
  • Ethanol (C2H5OH)
  • 13-nm Au colloid (synthesized in Steps 1–11 below)
  • Synthetic oligonucleotide probe for Au-NP with disulfide linker (HPLC purified)
  • SDS, Molecular biology grade (C12H25OSO3Na)
  • 30-nm Au-NPs (Ted Pella, Inc.)
  • Polyclonal antibody or second monoclonal antibody
  • Synthetic oligonucleotide probe with disulfide linker (HPLC purified)
  • Formamide, molecular biology grade (aliquot, store 4 °C) (CH3NO)
  • tRNA, 10,000 units (Sigma)
  • Tween 20 or Polysorbate 20, molecular biology grade (C58H114O26)
  • BSA (bovine serum albumin; Ambion)
  • Microarray slides spotted with capture oligonucleotide (4/10-well layout)
  • Silver enhancement solutions A/B (Nanosphere Inc. or Sigma)
  • Sodium nitrate, molecular biology grade (NaNO3)
  • Fluorophore reference standards
  • Fluorophore-labeled Au-NP oligonucleotide barcode

Equipment

  • 0.45-μm acetate filter (Millipore)
  • UV-visible spectrophotometer and cuvette
  • Drying oven
  • 500-ml volumetric flask
  • 50-ml volumetric flask
  • 1,000-ml two-neck round-bottom flask
  • Reflux condenser
  • Rubber septa
  • Tygon tubing
  • Glass pipettes or plastic spatula
  • Teflon tape
  • 1,000-ml all-glass filter holder apparatus
  • 500-ml amber-colored glass storage container
  • Transmission electron microscope (TEM) may also be required
  • Magnetic separators: 2 ml, 15 ml and 50 ml
  • Lyophilizer
  • Orbital, rocking or rotating shaker
  • 15- and 50-ml conical tubes
  • 1.5-ml microcentrifuge tubes
  • 10-ml syringes
  • 18-gauge needles
  • Aluminum foil
  • 20-ml clear borosilicate glass vials (conventionally known as EPA vials)
  • pH paper and pH meter
  • Eppendorf thermal mixer or temperature-controlled orbital shaker
  • VerigeneID (Nanosphere Inc) or traditional flatbed scanner
  • Hybridization gaskets, 4-well or 10-well layout (Nanosphere Inc)
  • Slide spinner for drying
  • Controlled-humidity chamber
  • Fluorescence plate reader or fluorometer
  • Fluorescence cuvette or 96-well Black/Clear bottom microtiter plate (Costar)
  • GenePix Pro (Molecular Devices), ImageQuant (GE Healthcare) or any other intensity quantification software, although GenePix is preferable

Reagent setup

  • 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.
  • Coupling buffer 100 mM phosphate buffer + 200 mM NaCl (pH = 6.9–7.0): 4.392 g Na2HPO4, 2.291 g NaH2PO4, 5.884 g NaCl, 500 mL NANOpure water.
  • Disulfide cleavage buffer 170 mM phosphate buffer (pH = 8.0): 11.468 g Na2HPO4, 0.509 g NaH2PO4, 500 ml NANOpure water.
  • Storage buffer 10 mM phosphate buffer + 200 mM NaCl (pH = 7.4): 0.570 g Na2HPO4, 0.118 g NaH2PO4, 5.844 g NaCl, 500 ml NANOpure water.
  • pH buffers 5 M NaOH and 5 M HCl prepared in NANOpure water.
    Caution Can cause burns.
  • Borate buffer 100 mM borate buffer (pH = 9.5): 3.091 g H3BO3, 500 ml NANOpure water.
  • Washing buffer 10 mM phosphate buffer + 150 mM NaCl (pH = 7.4): 0.562 g Na2HPO4, 0.125 g NaH2PO4, 4.383 g NaCl, 500 ml NANOpure water.
  • Passivation buffer 2 200 mM Tris (pH = 8.5): 1.210 g Tris, 50 ml NANOpure water.
  • Salting buffer 10 mM phosphate buffer + 2 M NaCl (pH 7.0): 0.0562 g 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 Na2HPO4, 0.125 g NaH2PO4, 50 ml NANOpure water.
  • Surfactant solution 10% SDS (wt/vol): 10 g SDS, 90 ml NANOpure water.
  • Bio-barcode assay components for protein and nucleic acid detection: There are many commercially available kits (e.g., Promega, Qiagen, Sigma) for the isolation of total DNA from soil, water, feces, tissue and blood, among other materials. As with any biological detection assay, sample preparation and handling are critical. All samples should be stored at 4 °C or lower if possible to prevent degradation from nucleases or proteases.
  • Assay buffer 10 mM phosphate + 150 mM NaCl + 0.1% SDS (wt/vol)(pH = 7.4): 0.562 g Na2HPO4, 0.125 g NaH2PO4, 4.383 g NaCl, 500 mL NANOpure water. For protein detection, omit SDS and include 0.1% BSA (wt/vol) and 0.025% Tween 20 (vol/vol).
  • Slide washing buffer A 0.5 M NaNO3 + 0.01% SDS + 0.02% Tween 20: 21.25 g NaNO3, 500 μl 10% SDS, 100 μl Tween 20, 500 ml NANOpure water.
  • Slide washing buffer B 0.5 M NaNO3: 21.25 g NaNO3, 500 ml NANOpure water.
  • Slide washing buffer C 0.1 M NaNO3 (store at 4 °C): 2.125 g NaNO3, 500 ml NANOpure water.

Equipment setup

  • Hybridization chamber for scanometric detection A hybridization chamber is required during the heated steps of the scanometric detection to prevent evaporation from the sample wells. The chamber can be easily made. Simply place 15 ml of the assay buffer into the bottom of an empty box used to hold pipette tips, set the slides on top of the empty tip rack and place the lid on top.
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Procedure

  1. 13-nm Au-NP synthesis. This procedure is adapted from ref. 16.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.

  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.
  12. Magnetic particle functionalization with DNARemove 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.
  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).
  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.
  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 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.
  43. Au-NP functionalization with DNALyophilize 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.)
    Figure 2: Testing antibody loading.
    Figure 2 : Testing antibody loading.

    The Au-NP–antibody loading test using salt to determine reasonable antibody amounts for probe generation. From these data, 6 μg ml−1 would be an appropriate amount of antibody to add, because the particles to the left have aggregated, and the ones to the right have not. The lack of aggregation indicates there is too much surface coverage to leave space for the thiolated oligonucleotide barcodes to attach. Image courtesy of Dr. A.-H. Bae.

    Full size image (40 KB)

  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.
  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.
  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.
  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.
  61. Bio-barcode assay for DNA detectionDetermine 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.
  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.Troubleshooting
    1. Scanometric detection of the barcodes (DNA and protein detection)
      1. 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.
      2. Incubate at 50 °C for 15 min, wash in NANOpure water and spin dry.
      3. Assemble the chip in the holder in the hybridization chamber.
      4. 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.
      5. Spin the supernatants for 5 min at 13,000g to pellet the aggregated gold particles.
      6. 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.
      7. 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.
      8. 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.
      9. 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.
      10. 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.
      11. 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.
      12. Fill each well with 15 μl universal probe solution from Step xi.
      13. Allow the probe to associate with the chip for 45 min at 37 °C in the controlled-humidity chamber with shaking at 120 r.p.m.
      14. Disassemble apparatus, and wash the slides individually twice in 40 ml of slide washing buffer A in a conical tube for 1 min.
      15. Next, wash the slides three times in 40 ml slide washing buffer B for 1 min at a time.
      16. Finally, quickly immerse the slides in 40 ml cold slide washing buffer C, and spin dry.
      17. Lay the slide flat on the benchtop on a folded Kimwipe.
      18. 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.

      19. Pipette silver stain onto slide, ensuring that the lower two-thirds of the slide are completely covered.
      20. 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.
      21. Wash slide thoroughly with NANOpure water, and spin dry.
      22. Image with Verigene-ID or conventional flatbed scanner, and save (Figs. 3 and 4).
        Figure 3: Scanometric image.
        Figure 3 : Scanometric image.

        This image shows how slides should look after silver enhancement as seen by the VerigeneID. The image shows the scattering of the silver-enhanced spots on the slide. Row 1, negative control; row 2, high-femtomolar target; row 3, mid-femtomolar target; row 4, low-femtomolar target.

        Full size image (14 KB)

        Figure 4: DNA detection image.
        Figure 4 : DNA detection image.

        This image shows how the data should look after running the bio-barcode assay and completing the silver enhancement as seen using a false coloring scheme with the GenePix software. Row 1, control; row 2, high-femtomolar target; row 3, mid-femtomolar target; row 4, low-femtomolar target.

        Full size image (14 KB)

      23. Quantify spot intensity using quantification software.
    2. Fluorescence detection of the barcodes (DNA and protein detection)
      1. After barcode release, extract the magnetic particles down on the magnet for 3 min and transfer the supernatants to clean microcentrifuge tubes.
      2. Spin the supernatants for 5 min at 13,000g to pellet the aggregated gold particles.
      3. Prepare serial dilution of known concentrations in barcode release buffer to generate a calibration curve.
      4. Remove supernatant from gold pellet and place in a new microcentrifuge tube.
      5. Adjust the volume of released barcodes to 100 μl using additional barcode release buffer.
      6. 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.
      7. Measure fluorescence and perform data analysis.Troubleshooting
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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)
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
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
Steps (ix)-(xii), 15 min
Step (xiii), 45 min
Steps (xiv)-(xxiii), 45 min
Total time ~4 h
(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

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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.

* 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.

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Acknowledgments

C.A.M. acknowledges HSARPA, NIH Pioneer Award, NIH NIAID, AFOSR, Doris Duke Charitable Foundation, NSF/NSEC and NCI. H.D.H: This work was performed while on appointment as a U.S. Department of Homeland Security (DHS) Fellow under the DHS Scholarship and Fellowship Program, a program administered by the Oak Ridge Institute for Science and Education (ORISE) for DHS through an interagency agreement with the U.S. Department of Energy (DOE). ORISE is managed by Oak Ridge Associated Universities under DOE contract number DE-AC05-06OR23100. All opinions expressed in this paper are the author's and do not necessarily reflect the policies and views of DHS, DOE or ORISE.

Competing interests statement: 

The authors declare no competing financial interests.

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References

  1. Nam, J.M., Thaxton, C.S. & Mirkin, C.A. Nanoparticle-based bio-bar codes for the ultrasensitive detection of proteins. Science 301, 1884–1886 (2003). | Article | PubMed | ISI | ChemPort |
  2. Nam, J.M., Stoeva, S.I. & Mirkin, C.A. Bio-bar code–based DNA detection with PCR-like sensitivity, J. Am. Chem. Soc. 126, 5932–5933 (2004). | Article | PubMed | ISI | ChemPort |
  3. Mirkin, C.A., Letsinger, R.L., Mucic, R.C. & Storhoff, J.J. A DNA-based method for rationally assembling nanoparticles into macroscopic materials, Nature 382, 607–609 (1996). | Article | PubMed | ISI | ChemPort |
  4. Storhoff, J.J. et al. What controls the optical properties of DNA-linked gold nanoparticle assemblies? J. Am. Chem. Soc. 122, 4640–4650 (2000). | Article | ISI | ChemPort |
  5. Storhofff, J.J., Elghanian, R., Mirkin, C.A. & Letsinger, R.L. Sequence-dependent stability of DNA-modified gold nanoparticles. Langmuir 18, 6666–6670 (2002). | Article |
  6. Jin, R.C. et al. What controls the melting properties of DNA-linked gold nanoparticle assemblies? J. Am. Chem. Soc. 125, 1643–1654 (2003). | Article | PubMed | ISI | ChemPort |
  7. Lytton-Jean, A.K.R. & Mirkin, C.A. A thermodynamic investigation into the binding properties of DNA functionalized gold nanoparticle probes and molecular fluorophore probes. J. Am. Chem. Soc. 127, 12754–12755 (2005). | Article | PubMed | ChemPort |
  8. Demers, L.M. et al. Thermal desorption behavior and binding properties of DNA bases and nucleosides on gold. J. Am. Chem. Soc. 124, 11248-11249 (2002).
  9. Thaxton, C.S. et al. A bio-bar-code assay based upon dithiothreitol-induced oligonucleotide release. Anal. Chem. 77, 8174–8178 (2005). | Article | PubMed | ChemPort |
  10. Demers, L.M. et al. A fluorescence-based method for determining the surface coverage and hybridization efficiency of thiol-capped oligonucleotides bound to gold thin films and nanoparticles. Anal. Chem. 72, 5535–5541 (2000). | Article | PubMed | ChemPort |
  11. Taton, T.A., Mirkin, C.A. & Letsinger, R.L. Scanometric DNA array detection with nanoparticle probes, Science 289, 1757–1760 (2000). | Article | PubMed | ISI | ChemPort |
  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). | Article | PubMed | ISI | ChemPort |
  13. Oh, B.K., Nam, J.M., Lee, S.W. & Mirkin, C.A. A fluorophore-based bio-barcode amplification assay for proteins, Small 2, 103–108 (2006). | Article | ChemPort |
  14. Stoeva, S.I. et al. Multiplexed detection of protein cancer markers with bio-barcoded nanoparticle probes, J. Am. Chem. Soc. (2006) doi: DI: 10.1020/ja0613106. | Article |
  15. Stoeva, S.I., Lee, J.-S., Thaxton, C.S. & Mirkin, C.A. Multiplexed DNA detection with biobarcoded nanoparticle probes. Angew. Chem. Int. Ed. Engl. 45, 3303–3306 (2006). | Article | PubMed | ChemPort |
  16. Frens, G. Controlled nucleation for regulation of particle-size in monodisperse gold suspensions. Nat. Phys. Sci. 241, 20–22 (1973).> | ChemPort |
  1. Northwestern University Department of Chemistry and The International Institute for Nanotechnology, 2145 Sheridan Road, Evanston, Illinois 60208, USA.

Correspondence to: Chad A Mirkin1 e-mail: chadnano@northwestern.edu

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