This document summarizes a new UV-visible spectroscopy method for quantifying the number and ratio of unlabeled DNA strands bound to gold nanoparticles (AuNPs) of different sizes. The method allows determining the number of both recognition and diluent DNA sequences on the AuNPs without using fluorescent labels. When applied to AuNPs of 5 nm and 12 nm, the method showed the ratio of DNA sequences bound was different for the different sized AuNPs, suggesting the AuNP radius of curvature influences DNA assembly.
A TaqMan-based Quantitative RT-PCR Method for Detection of Apple Chlorotic Le...Agriculture Journal IJOEAR
Abstract—ACLSV is one of the major fruit viruses and can cause severe diseases in species of family Rosaceae. Previous RT-PCR methods are available to detect ACLSV in hawthorn samples, but not to evaluate the infected level of ACLSV. In this study, a TaqMan-based quantitative RT-PCR detection method targeting CP gene of ACLSV was first established and the sensitivity and reproducibility were investigated. The results indicated that this standard curve between log of plasmid DNA concentration versus the cycle threshold (Ct) value generated a linear fit with a linear correlation (R2) of 0.99 and the PCR efficiency was more than 90%. The quantitative RT-PCR method was high sensitive and able to detect 6.9 × 102 copies•μL-1 of ACLSV RNA. Compared with the conventional RT-PCR method, it was 100-fold sensitive in detection of ACLSV. In addition, different organs of hawthorn samples were examined using the quantitative RT-PCR repeatedly and the result revealed that the quantitative RT-PCR is not only an effective detection method, and can obtain an absolute quantitation for ACLSV.
RNA Integrity and Quality – Standardize RNA Quality Control QIAGEN
RNA integrity and quality are critical to obtain meaningful and reliable downstream data. This slidedeck details the challenges and considerations of handling RNA samples, the need for quality control analysis and common methods for RNA integrity and quality assessment. The QIAxcel Advanced System will be introduced to automate the process of RNA sample integrity analysis and obtain objective quality measurement. Application data will be presented.
A TaqMan-based Quantitative RT-PCR Method for Detection of Apple Chlorotic Le...Agriculture Journal IJOEAR
Abstract—ACLSV is one of the major fruit viruses and can cause severe diseases in species of family Rosaceae. Previous RT-PCR methods are available to detect ACLSV in hawthorn samples, but not to evaluate the infected level of ACLSV. In this study, a TaqMan-based quantitative RT-PCR detection method targeting CP gene of ACLSV was first established and the sensitivity and reproducibility were investigated. The results indicated that this standard curve between log of plasmid DNA concentration versus the cycle threshold (Ct) value generated a linear fit with a linear correlation (R2) of 0.99 and the PCR efficiency was more than 90%. The quantitative RT-PCR method was high sensitive and able to detect 6.9 × 102 copies•μL-1 of ACLSV RNA. Compared with the conventional RT-PCR method, it was 100-fold sensitive in detection of ACLSV. In addition, different organs of hawthorn samples were examined using the quantitative RT-PCR repeatedly and the result revealed that the quantitative RT-PCR is not only an effective detection method, and can obtain an absolute quantitation for ACLSV.
RNA Integrity and Quality – Standardize RNA Quality Control QIAGEN
RNA integrity and quality are critical to obtain meaningful and reliable downstream data. This slidedeck details the challenges and considerations of handling RNA samples, the need for quality control analysis and common methods for RNA integrity and quality assessment. The QIAxcel Advanced System will be introduced to automate the process of RNA sample integrity analysis and obtain objective quality measurement. Application data will be presented.
Pcr technology and its importance in covid 19 pandemicAnupam Maity
Since the discovery of the PCR technology, its application in the various fields is increased gradually. Based on to this principle, many variations of the PCR have been established. Year by year, it is upgraded very much. It is established as a most common and accurate technique for the detection of the various diseases in the field of medicine. Now it is a ‘Gold standard’ for the detection of covid-19 also, which is much needed to contain the spread of the virus. Though various detection techniques are there for detection, but real time RT-PCR (variation of PCR) is most reliable. Viral detection is based on a simple principle of nucleic acid (viral) amplification. Various manufacturing companies are manufacturing the PCR instrument. Though the accuracy of the instruments are slightly differ to each other.
Currently, human papillomavirus (HPV) DNA tests validated
in large trials and epidemiological studies are the hybrid
capture second-generation (HC2) HPV DNA assay and
a variety of polymerase chain reaction (PCR) protocols employing
degenerate or consensus primers. This article describes
the currently available technology for HPV detection
and discusses novel technologies and their potential for
large-scale screening. Ideally, an HPV test should allow detection
of multiple HPV types, identify individual types, and
provide quantitative information about the viral load of each
individual type found. Moreover, it should be easy to perform,
be highly reproducible, with a high specificity and
sensitivity, and amenable for high throughput analysis and
automation. Because we do not yet fully understand the true
value of viral load and the biological relevance of the different
HPV types, any HPV test should be able to detect the
clinically relevant high-risk types with a sufficient sensitivity
of at least 10 000 genome copies per sample. To validate the
different current and future test systems and to compare
inter-laboratory performance we urgently need reference
samples, validated reagents, and standardized protocols.
Introduction to Real Time PCR (Q-PCR/qPCR/qrt-PCR): qPCR Technology Webinar S...QIAGEN
This slidedeck introduces the concepts of real-time PCR and how to conduct a real-time PCR assay. The topics that are covered include an overview of real-time PCR chemistries, protocols, quantification methods, real-time PCR applications and factors for success.
Sequencing is one of the major technological advancement that has taken shape in the last two or three decade. Starting from Sanger and Maxam-Gilbert sequencing methods to the latest high-throughput methods, sequencing technologies has changed the the landscape of biological sciences.
This slide takes a look a the major sequencing methods over time.
Note: Several images included here have been sourced from GOOGLE IMAGES. The content has been extracted from several SCIENTIFIC PAPERS and WEBSITES.
PLEASE DO CONTACT THE AUTHOR DIRECTLY IF ANY COPYRIGHT ISSUE ARISES.
Automated DNA purification from diverse Microbiome samples using dedicated Mi...QIAGEN
This application note demonstrates the automation of QIAGEN’s new line of DNA sample prep kits for the microbiome. The microbiome of samples as diverse as soil, water and stool was purified using dedicated QIAcube compatible kits. Automation on the QIAcube enabled efficient and reliable use of these samples for sensitive downstream applications such as qPCR and NGS. In addition, the CLC Microbial Genomics Module was successfully employed for metagenome sequencing and identification of microbial composition and diversity.
Modeling DNA Amplification by Polymerase Chain Reaction (PCR)Danielle Snowflack
The objective of this lesson is for students to gain hands-on experience of the principles and practice of Polymerase Chain Reaction (PCR). At the completion of this activity, students should understand the process by which PCR amplifies DNA.
Ribosomes are complex structures found in all living cells which functions in protein synthesis machinery. Basically ribosome’s consists of two subunits, each of which is composed of protein and a type of RNA, known as ribosomal RNA (rRNA). Prokaryotic ribosomes consist of 30S subunit (small sub unit) and 50S subunit (large sub unit) which together make up the complete 70S ribosome, where S stands for Svedberg unit non-SI unit for sedimentation rate. 30S subunit is composed of 16S ribosomal RNA and 21 polynucleotide chains while 50S subunit is composed of two rRNA species, the 5S and 23S rRNAs. The presence of hyper variable regions in the 16S rRNA gene provides a species specific signature sequence which is useful for bacterial identification process. 16S Ribosomal RNA sequencing is widely used in microbiology studies to identify the diversities in prokaryotic organisms as well as other organisms and thereby studying the phylogenetic relationships between them. The advantages of using ribosomal RNA in molecular techniques are as follows
Ribosomes and ribosomal RNA are present in all cells.
RNA genes are highly conserved in nature.
Culturing of microbial cells is absent in the sequencing techniques.
Pcr technology and its importance in covid 19 pandemicAnupam Maity
Since the discovery of the PCR technology, its application in the various fields is increased gradually. Based on to this principle, many variations of the PCR have been established. Year by year, it is upgraded very much. It is established as a most common and accurate technique for the detection of the various diseases in the field of medicine. Now it is a ‘Gold standard’ for the detection of covid-19 also, which is much needed to contain the spread of the virus. Though various detection techniques are there for detection, but real time RT-PCR (variation of PCR) is most reliable. Viral detection is based on a simple principle of nucleic acid (viral) amplification. Various manufacturing companies are manufacturing the PCR instrument. Though the accuracy of the instruments are slightly differ to each other.
Currently, human papillomavirus (HPV) DNA tests validated
in large trials and epidemiological studies are the hybrid
capture second-generation (HC2) HPV DNA assay and
a variety of polymerase chain reaction (PCR) protocols employing
degenerate or consensus primers. This article describes
the currently available technology for HPV detection
and discusses novel technologies and their potential for
large-scale screening. Ideally, an HPV test should allow detection
of multiple HPV types, identify individual types, and
provide quantitative information about the viral load of each
individual type found. Moreover, it should be easy to perform,
be highly reproducible, with a high specificity and
sensitivity, and amenable for high throughput analysis and
automation. Because we do not yet fully understand the true
value of viral load and the biological relevance of the different
HPV types, any HPV test should be able to detect the
clinically relevant high-risk types with a sufficient sensitivity
of at least 10 000 genome copies per sample. To validate the
different current and future test systems and to compare
inter-laboratory performance we urgently need reference
samples, validated reagents, and standardized protocols.
Introduction to Real Time PCR (Q-PCR/qPCR/qrt-PCR): qPCR Technology Webinar S...QIAGEN
This slidedeck introduces the concepts of real-time PCR and how to conduct a real-time PCR assay. The topics that are covered include an overview of real-time PCR chemistries, protocols, quantification methods, real-time PCR applications and factors for success.
Sequencing is one of the major technological advancement that has taken shape in the last two or three decade. Starting from Sanger and Maxam-Gilbert sequencing methods to the latest high-throughput methods, sequencing technologies has changed the the landscape of biological sciences.
This slide takes a look a the major sequencing methods over time.
Note: Several images included here have been sourced from GOOGLE IMAGES. The content has been extracted from several SCIENTIFIC PAPERS and WEBSITES.
PLEASE DO CONTACT THE AUTHOR DIRECTLY IF ANY COPYRIGHT ISSUE ARISES.
Automated DNA purification from diverse Microbiome samples using dedicated Mi...QIAGEN
This application note demonstrates the automation of QIAGEN’s new line of DNA sample prep kits for the microbiome. The microbiome of samples as diverse as soil, water and stool was purified using dedicated QIAcube compatible kits. Automation on the QIAcube enabled efficient and reliable use of these samples for sensitive downstream applications such as qPCR and NGS. In addition, the CLC Microbial Genomics Module was successfully employed for metagenome sequencing and identification of microbial composition and diversity.
Modeling DNA Amplification by Polymerase Chain Reaction (PCR)Danielle Snowflack
The objective of this lesson is for students to gain hands-on experience of the principles and practice of Polymerase Chain Reaction (PCR). At the completion of this activity, students should understand the process by which PCR amplifies DNA.
Ribosomes are complex structures found in all living cells which functions in protein synthesis machinery. Basically ribosome’s consists of two subunits, each of which is composed of protein and a type of RNA, known as ribosomal RNA (rRNA). Prokaryotic ribosomes consist of 30S subunit (small sub unit) and 50S subunit (large sub unit) which together make up the complete 70S ribosome, where S stands for Svedberg unit non-SI unit for sedimentation rate. 30S subunit is composed of 16S ribosomal RNA and 21 polynucleotide chains while 50S subunit is composed of two rRNA species, the 5S and 23S rRNAs. The presence of hyper variable regions in the 16S rRNA gene provides a species specific signature sequence which is useful for bacterial identification process. 16S Ribosomal RNA sequencing is widely used in microbiology studies to identify the diversities in prokaryotic organisms as well as other organisms and thereby studying the phylogenetic relationships between them. The advantages of using ribosomal RNA in molecular techniques are as follows
Ribosomes and ribosomal RNA are present in all cells.
RNA genes are highly conserved in nature.
Culturing of microbial cells is absent in the sequencing techniques.
In this presentation you will get a deep insight on the most important step of DNA fingerprinting that is the Quantitation of DNA.
You will understand what is DNA quantitation and also about the different techniques of DNA quantitation.
This powerpoint explains about the nucleic acid hybridization, its principle, application and the assay methods. Also it gives clear picture about DNA probes, its sysnthesis, mechanism of probes and the detector system in DNA hybridization.
WHAT IS BLOTTING?
Blotting is a technique for detecting any macromolecules that we deal with like DNA, RNA or proteins, which are initially present in a complex mixture.
TYPES OF BLOTTING:
Southern Blotting
Northern Blotting
Western Blotting
NORTHERN BLOTTING
A northern blotting is a laboratory method used to detect specific RNA molecules among a mixture of RNA (mRNA).
The technique was developed in 1979 by James Alwine and his colleagues.
Northern blotting can be used to analyze a sample of RNA from a particular tissue or cell type in order to measure the expression of particular genes.
Northern blotting involves the use of electrophoresis to separate RNA samples by size, and detection with a hybridization probe complementary to part of or the entire target sequence.
The term ‘northern blot’ actually refers specifically to the capillary transfer of RNA from the electrophoresis gel to the blotting membrane. However the entire process is commonly referred to as northern blotting.
PROCEDURE
1.RNA isolation:
2.Separation of RNA using gel electrophoresis:
3.BLOTTING:
4.Hybridization with labelled probe:
5.WASHING OFF EXCESS PROBES
4. (TCO 9) Provide a detailed description of the techniques used to .pdfarrowit1
4. (TCO 9) Provide a detailed description of the techniques used to make a DNA fingerprint.
What are some of the uses and applications of DNA fingerprinting?
Solution
DNA fingerprinting is a technique used to determine the nucleotides sequences of DNA which
are unique to each individual.
Technique
1. Extraction of the DNA from the source the DNA is extracted from blood sample, hair follicles
etc.available sample.
2.DNA is cut into fragments the DNA molecules broken with the help of restriction
endonuclease. Here the cleaning is double strand cut producing DNA fragments of different
lengths this fragment are also called restricted fragment length polymorphism Manyi of this
fragment contain vntr
3. Separation of the fragments using gel electrophoresis. As the DNA molecule is negatively
charged hence it will move towards positive or not in the setup the gel based matrix provides tiny
pores through which DNA molecules travel the larger molecules travel slowly where is the
smallest mens travel quickly from the loading point at the end of the experiment DNA pieces of
equal length obtained.
4. The DNA fragments or now treated with alkaline chemicals to facilitate denaturation into
single stranded DNA this is very important step.
5. Southern blotting technique in this technique nitrocellulose membrane is used the DNA is
bloated on suitable membrane like nitrocellulose or nylon membranes as they have good binding
capacity the membrane is subjected to gentle pressure due to this single stranded DNA fragments
are pulled and transfer onto the membrane . the membrane contains replica of the DNA.
Hybridisation with suitable DNA probe which is single stranded DNA having complementary
sequence to the desired DNA. Before using the probe the DNA of tagged with fluorescent dyes
to help in detection of the desired DNA excess probea are washed away.
5. the DNA sample is visualised using autoradiography the hybridisation pattern is called DNA
fingerprint having a sequence complementary to the probe.
6. PCR technique is a technique is useful to synthesise millions of copies of the DNA sequence
when low amount of DNA is available for the study this technique is used modifications of PCR
technique like r a p d PCR rflp PCR helps in giving accurate results.
Applications of DNA fingerprinting :
1.This test is used in the case of disputes regarding paternity testing .
2 it is useful tool in forensic applications
3.It is used to assess migration pattern of ancient population
4.it is used to determine Genetic diversity is in the evolutionary biology.
4. It is used to diagnose inherited disorders in both prenatal and newborn babies examples
huntington\'s disease Alzheimer\'s Sickle Cell anaemia Thalassemia haemophilia.
5. DNA fingerprinting is used to come from confirm cell line identity in a cell line collection.
6. It also helps in developing cures for inherited disorders..
INTRODUCTION
Hybridization stages
probe synthesis
Probe marking
Target DNA processing
Target DNA denaturation
Target DNA transfer to solid carrier
Visualization
CONCLUSIONS
REFERENCES
2. displaced DNA is quantified by fluorescence emission. In the
“turn-off” method, the fluorescence emission of a fluorophore-
labeled DNA solution is determined before and after incubation
with AuNPs, and the DNA concentration is quantified from the
decrease in fluorescence due to quenching by the AuNPs. In
both cases, the concentration of AuNPs is determined from
their UV−vis absorbance at 520 nm.
The main drawbacks of the “turn-on” and “turn-off”
fluorescence quantification methods are that each DNA
sequence to be quantified must be labeled with a different
fluorophore, and assumptions must be made about the
interactions between ligands and the AuNP core. Fluorophore
labels can affect DNA−NP ligand structure, reactivity, and the
number of recognition strands bound to each AuNP.14,27
They
are also time-consuming to synthesize, expensive to purchase,
and often bleach under light exposure.14
To determine the
DNA concentration using the fluorescent turn-on method, it
must be assumed that all DNA ligands are completely displaced
from the surface of the DNA−NPs. This can be problematic
because the rate and extent of thiol/thiol ligand exchange
depends strongly on the ligand identity.28
To determine the
DNA concentration using the fluorescent turn-off method, it
must be assumed that the fluorophore is completely quenched
upon interacting with the AuNPs.29
Label-free DNA sequences attached to AuNPs have been
quantified using the Oligreen fluorescence assay15,30
and the
toehold displacement assay.14
The Oligreen assay is suitable for
quantifying DNA in solutions containing sequences longer than
six nucleotides,30
but is restricted to quantifying one DNA
sequence. The toehold displacement assay is suitable for
simultaneous quantification of recognition and diluent
sequences, but only for sequences longer than 18 nucleotides.14
Both methods require specialized equipment and reagents and
assume complete ligand displacement by small thiol molecules.
To date, no method exists to determine the number of DNA
strands per AuNP without using fluorophores or to quantify
recognition and diluent ligands in the same sample.
New methods to determine the number of DNA strands per
AuNP without using fluorophores or making assumptions
regarding nanoparticle reactivity would be useful for quantifying
both the recognition and diluent DNA strands within a given
sample. A current barrier to label-free detection using UV−vis
spectroscopy is that AuNPs and their common impurities
absorb light at the wavelength typically used for DNA detection
(260 nm). We found that the contribution from these species
can be subtracted after KCN digestion of the nanoparticles.
In this paper, we describe a convenient, inexpensive UV−vis-
based method to quantify the number of DNA strands bound
to AuNPs. This method is suitable for determining the number
of DNA strands bound to gold nanoparticles at typical
nanoparticle working concentrations (e.g., 5−50 nM for 12
nm AuNPs). Using this method in conjunction with a
commercially available dye assay, it is possible to determine
the number of recognition and diluent DNA sequences bound
to AuNPs, allowing us to determine their ratio as a function of
the feed ratio during ligand exchange. We demonstrate that this
method can be applied to large (12 nm) and small (5 nm)
AuNPs. The results of determining the number of recognition
and diluent strands bound to small versus large AuNPs suggest
that the AuNP radius of curvature has a large influence on DNA
assembly onto the AuNPs.
■ EXPERIMENTAL SECTION
Materials and Reagents. Citrate-stabilized AuNPs (dcore =
5 nm) were purchased from Nanocomposix (San Diego, CA).
All DNA samples were purchased from Integrated DNA
Technologies (Coralville, IA). DNA sequences were purified by
either the standard desalting method or HPLC. “Quant-It”
OliGreen ssDNA Assay kits were purchased from Thermo
Fisher Scientific (Grand Island, NY). The 50 kDa spin column
purification membranes were purchased from Millipore
(Darmstadt, Germany). Clear and amber 1.5 mL micro-
centrifuge tubes and opaque polypropylene black 96-well plates
(Costar) were purchased from VWR (Radnor, PA). Sodium
citrate dihydrate, hydrogen tetrachloroaurate hydrate, and UV-
transparent 96-well plates (Corning) were purchased from
Sigma-Aldrich (St. Louis, MO).
Instrumentation. UV−vis absorbance spectra of AuNP and
DNA solutions were obtained using either a BioTek Synergy 2
instrument or a Mikropack DH-2000 UV−vis−NIR light
source equipped with an Ocean Optics USB2000 spectropho-
tometer. UV−vis spectra of DNA were obtained using these or
a Thermo Scientific Nanodrop 2000 spectrophotometer path
length (10 mm) and baseline-corrected at 340 nm.
The end point (after 5−15 min) fluorescent emission of the
solutions was measured in 96-well opaque black well plates
(Costar) using a Biotek Synergy 2 instrument equipped with a
tungsten lamp and filters (EX 485/20 nm, EM 528/20 nm).
The data collection time was autoscaled so that 80 000 counts
were emitted from the well containing the highest concen-
tration of DNA.
Calculation of UV−Visible Extinction Coefficients.
AuNP extinction coefficients were calculated using their average
core diameters (dcore = 12.3 nm, ε = 1.98 × 108
; dcore = 5.0 nm,
ε = 9.96 × 106
) and previously reported empirical data.31
The
error associated with these extinction coefficient values is 1−
3%.31
DNA sequence extinction coefficients (ε) were calculated
using Integrated DNA Technologies’ “Oligo Analyzer” tool,
which calculates values from thermodynamic modeling
according to DNA base composition and nearest neighbors
(Table 1).32
DNA extinction coefficient values are accurate
within 4% error (IDT-DNA). Concentrations of AuNP
solutions were determined from A520, and DNA concentrations
were determined from A260.
Preparation of 12 nm DNA−NPs. The 12 nm citrate-
stabilized AuNPs were synthesized using a modified literature
method.33,34
Briefly, a 250 mL three-neck round-bottom flask,
glass stopper, magnetic stir bar, and condenser were cleaned
using aqua regia and rinsed copiously with Nanopure water.
Sodium citrate dihydrate (408 mg, 1.39 mmol) was dissolved in
Table 1. Names, Primary Sequences, and Calculated
Extinction Coefficients of DNA Sequences Useda
name DNA primary sequence
extinction coefficient
(L mol−1
cm−1
)
DNA1 5′-AGA GAA CCT GGG GGA GTA
TTG CGG AGG AAG GT-3′
331 900
DNA2 5′-A5-3′ 63 400
DNA3 5′-A12-3′ 147 400
DNA4 5′-T5-3′ 41 100
DNA5 5′-CCC AGG TTC TCT-3′ 102 500
a
All sequences are labeled at their 5′ end with disulfide (HO-
(CH2)6S−S−5′-DNA-3′).
Analytical Chemistry Article
DOI: 10.1021/acs.analchem.6b02640
Anal. Chem. 2016, 88, 12072−12080
12073
3. 200 mL of Nanopure water and brought to 100 °C while
stirring. HAuCl4 (1 mL of 200 mM solution) was added using a
micropipettor. The solution instantly turned dark blue, a color
change previously attributed to nucleation.33
Within 1 min, the
solution turned a deep red color, indicating AuNPs were
formed. AuNPs were stirred at 100 °C for 20 min, then
removed from heat and allowed to stir overnight before being
characterized using small-angle X-ray scattering (SAXS) and
transmission electron microscopy (TEM).
The AuNP size determined by SAXS analysis was 12.3 ± 1.9
nm (Figure S-1). TEM analysis confirmed that the AuNPs were
spherical (Figure S-2). Descriptions of SAXS and TEM data
acquisition methods are available in the Supporting Informa-
tion.
AuNPs were functionalized with DNA using a modified
literature method.25,35
Typically, AuNPs and DNA were mixed
together to prepare reaction solutions containing 16 nM
AuNPs and 16 μM DNA. 10× excess DNA was added to
maximize DNA loading on the AuNPs, because a small but
measurable increase in DNA density during functionalization
was reported when excess DNA was used during ligand
exchange.35
After 5 min, pH 3 citric acid buffer was added (10
mM). After 10 min, NaCl was added (70 mM).
DNA and AuNPs were incubated overnight before being
purified using four rounds of centrifugation (15 min at 20
000g). DNA−NPs were redispersed in buffer containing 1 mM
pH 8.2 Tris acetate and 100 mM NaCl after each centrifugation
step, and finally dissolved in 225 μL of Nanopure water.
Fluorescence spectroscopy was used to determine that this
method removed all excess DNA (Figure S-3a). After each
purification, UV−vis spectroscopy was used to confirm that
most excess DNA (>99%) is removed.
Preparation and Analysis of 5 nm DNA−NPs. The same
procedures (with modifications) were used to prepare DNA−
NPs from purchased 5 nm AuNPs (Nanocomposix, San Diego)
and to analyze their ligand shell composition. SAXS analysis
confirmed the AuNPs were 5.0 ± 0.5 nm, and TEM confirmed
they were spherical. AuNPs and DNA were mixed together.
The reaction solutions contained 90 nM AuNPs and 13.5 μM
DNA. DNA−NPs were purified by centrifuging five times (9
min at 13 500g) above a spin filter membrane with a 50 kDa
molecular weight cutoff, discarding each flow-through.
Fluorescence spectroscopy was performed to determine that
this method removed all excess DNA (Figure S-3b). DNA−
NPs were eluted according to the manufacturer’s instructions
and redispersed using 230 μL of Nanopure water.
UV−Visible Spectroscopy Determination of DNA
Strands per Nanoparticle. The number of DNA strands
per AuNP was calculated by dividing the DNA concentration
by the AuNP concentration. The concentration of DNA−NPs
in each sample was determined using the A520 and calculated
extinction coefficient of AuNPs of the same core size. The
concentration of DNA in each sample was determined using
the A260 and calculated extinction coefficient of the DNA
sequence.
KCN solution (100 mM) was prepared in Nanopure water
adjusted to pH 12 using NaOH. This solution was mixed with
12 nm AuNPs or DNA−NPs and allowed to react overnight
before measuring the resultant UV−vis absorbance spectrum.
At minimum, 8 mol of KCN (4 equiv) was added for every
mole of Au atoms (15 mM, typically). The number of gold
atoms in solution was determined by calculating the number of
gold atoms per AuNP from the average AuNP volume and the
density and molar mass of gold, and multiplying it by the
number of nanoparticles in solution (calculated by multiplying
the solution volume, AuNP concentration, and Avogadro’s
number). The DNA A260 was determined by subtracting the
contribution of decomposed AuNPs from the A260 of the
DNA−NP decomposition reaction solution. The concentration
of DNA in each sample was determined based on its extinction
coefficient and DNA A260.
To validate our method, Quant-It’s ssDNA Oligreen
quantification assay30
was used to determine the DNA in the
decomposed AuNP solutions, using the supplier’s instructions.
Briefly, a series of standard DNA solutions (80, 40, 20, and 8
nM) were prepared. The decomposed DNA−NP A260 was used
to determine how much to dilute the decomposed DNA−NP
samples for the Oligreen assay. Buffer (pH 7.5, 10 mM Tris−
HCl, 1 mM EDTA) and water were added to each sample,
followed by the Oligreen dye. Samples were incubated for 5−10
min before measuring the final fluorescent emission of the dye.
The decomposed AuNPs did not affect the assay results.
To determine the ligand shell composition of the 5 nm
DNA−NPs, the same procedure was followed, except 2.5 mol
of KCN (1.25 equiv) was added for every mole of Au atoms
(typically 2−3 mM).
UV−Visible and Fluorescence Spectroscopy Determi-
nation of Two Types of DNA Sequences Bound to Gold
Nanoparticles. The number of strands of each DNA per
AuNP was determined by dividing the concentration of each
DNA sequence by the concentration of AuNPs. The
concentration of DNA−NPs was determined as described
previously. The concentration of the longer DNA strand was
determined using the Oligreen dye assay and used to calculate
the number of DNA strands per AuNP. The extinction
coefficient of the longer DNA strand was then used to
determine its contribution to A260 decomposed DNA−NPs.
The absorbance of the shorter DNA strand was then calculated
by subtracting the contributions from the decomposed
nanoparticles and the longer DNA strand from A260
decomposed DNA−NPs, and using the DNA’s extinction
coefficient to calculate its concentration. The percentage of
each strand in the ligand shell was then calculated.
■ RESULTS AND DISCUSSION
A simple approach to determining the number of DNA strands
per AuNP would be to measure the UV−vis spectrum of the
DNA−NPs and use the absorbances due to the gold cores and
the DNA bases to determine their concentrations. The
concentration of AuNPs can be determined using UV−vis
spectroscopy, based on their absorbance at 520 nm (A520) and
empirically determined extinction coefficients.31
AuNP ex-
tinction coefficients do not significantly change upon
functionalization with DNA.18
Therefore, established extinction
coefficient values could also be used to determine the
concentration of DNA−NPs. DNA concentrations are
conveniently determined from their absorbance at 260 nm
(A260) and extinction coefficients calculated using thermody-
namic modeling.32
The main reason UV−vis spectroscopy has not been used to
determine the DNA bound to AuNPs is that AuNPs and other
gold-containing impurities (gold salts and small gold clusters)
absorb light at 260 nm, the wavelength where DNA absorbs
most strongly.36
Therefore, the gold-based contributions
cannot be independently determined and subtracted from the
UV−vis spectra of intact DNA−NPs. In addition, gold-
Analytical Chemistry Article
DOI: 10.1021/acs.analchem.6b02640
Anal. Chem. 2016, 88, 12072−12080
12074
4. containing impurities present in the citrate-stabilized AuNPs
cannot be removed without destabilizing the NPs, making it
more difficult to determine the contribution of the AuNP cores
to A260 (prior to functionalization).
To eliminate the strong absorbance from the AuNP core, we
thought it would be feasible to decompose purified DNA−NPs
by treatment with cyanide prior to determining the DNA
concentration. Cyanide etching has long been used to extract
gold from ores and has previously been used to decompose
gold nanoparticles for quantification of DNA ligand shells
based on fluorescence18
and radioactivity.37
Our initial strategy to quantify the number of DNA strands
per AuNP was to determine the concentration of the DNA−
NPs from their A520, decompose the DNA−NPs using KCN
(Figure 1), and quantify the DNA based on A260. DNA−NPs
were prepared using an established method35
and purified by
centrifugation (Figure S-3) in dilute buffer before being
characterized using UV−vis spectroscopy. Buffers commonly
used to purify DNA−NPs absorb light at 260 nm (Figure S-4)
and must be diluted for accurate DNA quantification using this
method.
The solution of DNA−NPs (Figure 2a) exhibited a UV−vis
absorbance peak characteristic of AuNPs at 520 nm and an
absorbance peak characteristic of DNA at 260 nm. The latter
absorbance was absent from the UV−vis spectrum of DNA-free
AuNPs of the same core size, which was normalized to have the
same A520.
To use A260 to determine the DNA concentration, we
decomposed the DNA−NPs and AuNPs using KCN and then
measured the UV−vis spectra of the resultant solutions (Figure
2b). The decomposed DNA−NPs absorbed strongly at 230,
240, and 260 nm. (Figure 2b). The peaks at 230 and 240 nm
are characteristic of the KAu(CN)2 salt formed during
nanoparticle decomposition.38
The peak at 260 nm corre-
sponds to the DNA absorption.
The baseline absorbance of the solution containing KCN and
AuNPs or DNA−NPs approached zero at 350 nm, and the
spectrum did not change after 15 min or 12 h, respectively.
Therefore, all nanoparticles were allowed to react with cyanide
until their A350 approached zero. It is essential to evaluate the
end point of the reaction using UV−vis spectroscopy because
the reaction solutions appeared colorless by eye before the
spectrum stopped changing.
To determine the DNA concentration from A260, the
overlapping contribution from KAu(CN)2 is subtracted.
Because the absorbance due to KAu(CN)2 at 260 nm results
from the reaction of KCN and AuNPs, the absorbance can be
directly related to the initial AuNP concentration. Solutions
containing different concentrations of AuNPs were allowed to
react with KCN, and their UV−vis absorbance spectra
measured. A260 of the resultant solutions directly correlated
with the initial AuNP concentration (Figure 3a). A plot relating
A260 of the decomposed AuNPs to the initial AuNP
concentration (Figure 3b) was prepared. The fact that the
plot was linear meant this could be used as a calibration curve
to predict A260 for unknown solutions of DNA−NPs.
UV−Visible Spectroscopy Determination of DNA
Bound to AuNPs. To evaluate whether UV−vis spectroscopy
could be used to determine the number of DNA strands per
AuNP, we prepared AuNPs functionalized with DNA1 (5′-AGA
GAA CCT GGG GGA GTA TTG CGG AGG AAG GT-3′),
determined the DNA−NP concentration from A520, and used
eq 1 to determine A260 of DNA1, where dDNA−NPs denotes
decomposed DNA−NPs and dAuNPs are decomposed citrate-
stabilized nanoparticles. DNA1 was chosen as a representative
sequence for this study because its length (32 bases) is
comparable to recognition sequences typically used to
functionalize AuNPs. Using DNA1’s extinction coefficient
(Table 1) and Beer’s law, we determined that there were 58
± 7 DNA strands per 12 nm AuNP (n = 9).
= − −A A ADNA dDNA NPs dAuNPs260 1 260 260 (1)
Figure 1. Overall strategy for quantifying DNA bound to AuNPs using
UV−vis spectroscopy. (1) The concentration of DNA−NPs is
determined from their absorbance at 520 nm, (2) DNA−NPs are
decomposed using KCN, and (3) the concentration of DNA is
determined from the absorbance of the resultant solution at 260 nm,
as shown in the equation.
Figure 2. UV−vis absorbance spectra of solutions containing AuNPs
(a) before and (b) after oxidative KCN decomposition (arbitrary
units): (a) intact 12 nm DNA−NPs (solid line) and citrate-stabilized
AuNPs (dashed line); (b) products from reaction between 12 nm
DNA−NPs and KCN (solid line), AuNPs and KCN (dashed line).
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5. If A260 of the decomposed AuNPs was not subtracted from A260
of the solution prior to calculating the DNA concentration, it
would cause 28 ± 4% determinate error.
Method Validation. To validate this method, we also
quantified the number of DNA1 strands per AuNP using an
established Oligreen fluorescent dye assay.14,15,30,39,40
The value
calculated using this method (59 ± 4) agreed with our method
within 1% error, which is within the experimental error
introduced by determining the AuNP concentration using UV−
vis spectroscopy. This suggests that UV−vis spectroscopy can
be used to easily determine DNA bound per AuNP.
This method is advantageous because it is convenient,
inexpensive, and does not require using fluorophore-containing
reagents or making assumptions about ligand displacement
rates. Because extinction coefficients can be determined for any
DNA sequence, our method can be used to quantify shorter
DNA sequences than those analyzed using the Oligreen
fluorescence assay30
or toehold displacement assay.14
The
method is sufficiently sensitive to determine the number of
DNA strands bound to gold nanoparticles at typical DNA−NP
working concentrations (e.g., 5−50 nM).
The number of DNA1 (a 32 base sequence) strands per 12
nm AuNP (59 per AuNP) was lower than the number of DNA
(a 12 base sequence) strands adsorbed to similarly sized (13
nm) AuNPs (85 per AuNP) under similar conditions.35
After
taking into account the difference in surface areas between 12
and 13 nm AuNPs, the surface density of bound DNA1 strands
was 27% lower than the density reported for the shorter
sequence.35
The differences in density suggest the two DNA
sequences interact differently with the AuNP surface.
On the basis of these observations and those reported in the
literature, it seems likely that part of DNA1 lies flat on the
surface of the AuNPs during functionalization. Hurst et al.41
observed that the final surface coverage of thiolated DNA on
AuNPs is inversely related to the adenosine content of the
sequence near the thiol anchoring group, suggesting that bases
near the thiol anchoring group continue to lie flat and interact
strongly with the gold surface after the AuNPs are saturated
with DNA. Additional DNA binding took place when
interactions with the surface were disrupted by sonication.41
If DNA1 initially lies flat on the surface of our AuNPs, it may
electrostatically or sterically hinder the adsorption of additional
DNA, leading to the lower number of strands per AuNP.
UV−Visible and Fluorescence Spectroscopy Determi-
nation of Two Types of DNA Sequences Bound to Gold
Nanoparticles. Given the importance of using mixed and
diluted DNA ligand shells to control DNA−NP reactivity, we
wanted to extend our technique to determine the number of
recognition and diluent DNA sequences bound to the surface
of AuNPs. We prepared DNA−NPs functionalized with two
DNA sequences, DNA1 and DNA2 (5′-A5-3′), by performing
ligand exchanges on 12 nm AuNPs using solutions containing
DNA1 and DNA2. DNA2 was selected as a representative
diluent sequence because sequences of this length (≤5 bases)
do not interact appreciably with the Oligreen dye reagent.30
Typically, such short sequences must be labeled with a
fluorophore for quantification.
Determining the concentration of each sequence and
dividing it by the AuNP concentration (Figure 4) allowed the
percentage of DNA1 in the ligand shell to be determined.
The concentration of DNA−NPs was determined using
UV−vis spectroscopy, and the longer sequence, DNA1, was
determined using the Oligreen dye assay. The DNA1
concentration and its extinction coefficient were used to
determine its A260. The DNA2 concentration was then
determined from the absorbance of the decomposed DNA−
NP solution at 260 nm, after subtracting the contributions from
KAu(CN)2 and DNA1. The indeterminate error associated with
Figure 3. (a) Representative UV−vis spectra of solutions prepared by
reacting various concentrations (3.5−14.2 nM) of 12 nm AuNPs with
KCN. UV−vis spectroscopy can determine the DNA bound to AuNPs
in solutions containing ≥1.5 nM 12 nm AuNPs. (b) Calibration curve
used to determine the contribution of decomposed AuNPs to
decomposed DNA−NP absorbance spectra (n = 24). The R2
value
for the linear fit was 0.968.
Figure 4. UV−vis spectroscopy and fluorescent spectroscopy
determination of two different DNA strands (DNA1 and DNA2)
bound to AuNPs. (1) The concentration of DNA−NPs is determined
from A520, and used to determine A260 of KAu(CN)2. (2) DNA−NPs
are decomposed using KCN. (3) A260 of the resultant solution is
measured. (4) The concentration of DNA1 is determined from a linear
(typical R2
= 0.999) calibration curve relating DNA1 concentration to
Em528 and used to calculate A260 of DNA1. A260 of DNA2 is determined
by subtracting A260 of DNA1 and A260 of KAuCN2 from A260 of the
solution. The DNA2 concentration is then calculated using its
extinction coefficient.
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6. determining the percentage of DNA1 in the ligand shell was
2.6%, which reflects the variance between A260 of DNA1
determined from UV−vis versus fluorescence spectroscopy.
To use the Oligreen dye assay to measure DNA1 in solutions
containing DNA2, it must be assumed that DNA2 does not
appreciably affect the fluorescent emission of the assay solution.
To test this assumption, DNA1 was quantified using the
Oligreen dye assay with and without other DNA sequences
(DNA2, DNA3, or DNA4) present. In all cases, the shorter
DNA sequence did not affect the fluorescent emission of DNA1
(Figure S-5).
To use this method to quantify two different DNA sequences
bound to the nanoparticles, the sequences must exhibit greatly
different reactivity toward a commercially available fluorescent
dye, e.g., vary significantly in length or thymine composition.
To quantify two sequences of similar length, our method could
be used concurrently with a different technique. For mixtures of
sequences that are longer than 18 nucleotides, one ligand can
be quantified using our method and the other using sequential
strand displacement by DNA “toehold sequences”.14
From the results obtained by quantifying the number of
DNA1 and DNA2 strands bound per AuNP, it was apparent
that DNA1 was under-represented in the AuNP ligand shell
after functionalization (Figure 5). When equimolar amounts of
DNA1 and DNA2 were added during ligand exchange, only 3.5
± 0.1% of the DNA bound to the AuNP surface was DNA1.
This was intriguing because previous studies found a propor-
tional relationship between the feed ratio and bound DNA
ratio.18,25
To further investigate the impact of the ligand exchange feed
ratio on the type and number of DNA strands bound per
nanoparticle, we varied the ratio of recognition/diluent strands
during ligand exchange and used this method to quantify both
strands. In all cases, a lower percentage of DNA1 was present in
the AuNP ligand shell than was present in the ligand exchange
mixtures (Figure 5). There was a nonlinear relationship
between the percentage of DNA1 during ligand exchange and
the percentage of DNA1 bound to the AuNPs (Figure 5).
The reason that DNA1 is under-represented in the ligand
shell is likely because the DNA1 sequence is substantially longer
and less adenosine-rich. It has been shown that adsorption rate
of unthiolated42
and thiolated43
DNA sequences to AuNPs is
inversely related to the chain length of the sequence and that
the initial rate of DNA adsorption is directly related to the base
content of the sequence, with polyadenosine sequences
exhibiting the highest adsorption rate.44
The sequences used
in studies where the bound ratio was proportional to the feed
ratio were the same25
or similar length,18
with similar base
content near the anchoring thiol group, which explains why
they observed a linear proportional relationship between the
feed ratio and the bound DNA ratio.
Having investigated the effect of the feed ratio upon the
bound DNA ratio, we proceeded to investigate the effect of the
nanoparticle’s radius of curvature on the bound DNA ratio.
While the total number of thiolated DNA strands bound to an
AuNP increases as a function of its core size,41
the density of
bound DNA strands is inversely related to the nanoparticle’s
radius of curvature, with DNA strands forming a smaller
effective footprint on smaller AuNPs.39
We hypothesized that
the smaller effective footprint of the DNA ligands on a small
nanoparticle would allow the bulkier DNA1 ligand to make up a
larger proportion of the ligand shell.
Determination of Label-Free DNA Sequences Bound
to 5 nm AuNPs. To evaluate the effect of the nanoparticle
radius of curvature upon the bound DNA ratio, we prepared 5
nm nanoparticles functionalized with a mixture of recognition
(DNA1) and diluent (DNA2) sequences and used this analytical
method to determine the type and number of DNA strands
bound.
Five nanometer DNA−NPs were selected as a representative
size for this study because they are often used for fundamental
and applied studies.45−47
They are convenient for studying
assembly of DNA−NPs in solution, because their assemblies
are less prone to precipitation and therefore produce more
uniform SAXS patterns than DNA−NPs with larger core
sizes.45
Small AuNPs are advantageous for in vivo bioimaging
and drug delivery applications involving negatively charged
nanoparticles because of their increased propensity to enter
tumor cells.46,47
Solutions containing different concentrations of 5 nm citrate-
stabilized AuNPs were prepared, and the nanoparticles were
decomposed, and their UV−vis spectrum was measured. The 5
nm AuNPs contain fewer gold atoms than 12 nm AuNPs, and
they exhibited a lower A260 when decomposed. A260 of the
decomposed 5 nm DNA−NP solutions varied linearly with the
original NP concentration (Figure 6a).
The 5 nm DNA−NPs were prepared by incubating AuNPs
with DNA1, and the number of DNA1 strands per 5 nm AuNP
(n = 9) was analyzed using spectroscopy (18 ± 2) and the
fluorescent dye assay (15.1 ± 0.8). The calculated ranges of
DNA per AuNP agreed reasonably well, suggesting that the
UV−vis-based method is suitable for determining the DNA
bound to smaller, as well as larger, core sizes.
Although the primary focus of this work is smaller AuNPs
that exhibit desirable optical properties and high colloidal
stability, this method could also be extended toward quantifying
ligands bound to larger AuNPs. On the basis of the extinction
coefficient of DNA1, and the average number of DNA strands
that larger AuNPs bind,35,41
this method could be used to
determine the DNA bound to AuNPs in solutions containing
≥0.5 nM 50 nm AuNPs.
Figure 5. Ligand shell composition of DNA−NPs prepared by mixing
12 nm AuNPs with various amounts of DNA1 and DNA2 sequences.
The percentage of DNA1 in the ligand shell was determined using
UV−vis and fluorescence spectroscopy. Error bars represent the
standard deviation for the mean % DNA1 in ligands bound to 12 nm
AuNPs (error bars do not overlap).
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7. For AuNPs functionalized with mixtures of DNA1 and
DNA2, DNA1 was under-represented in the ligand shell of the 5
nm DNA−NPs, similar to what was observed for the larger
DNA−NPs. However, the ratio of DNA sequences bound to
the AuNPs was different for the large and small AuNPs. For
example, when AuNPs are incubated with solutions containing
75% DNA1 during ligand exchange, the ligand shell of the 12
nm nanoparticles contains 15 ± 3% DNA1 and the ligand shell
of the 5 nm nanoparticles contains 23 ± 3% DNA1.
This evidence that the ratio of DNA sequences bound to the
AuNPs was different for the large and small AuNPs suggested
that the radius of curvature influenced the assembly of DNA
strands onto the AuNPs. If chain length and adenosine content
were the only factors influencing DNA adsorption, AuNPs of
different core sizes functionalized using the same feed ratios of
DNA sequences would produce DNA−NPs with the same
ligand shell composition. Instead, the adsorption of longer
DNA sequences is promoted by increasing the AuNP radius of
curvature, which suggests a more complex reaction mechanism.
The influence of the nanoparticle’s radius of curvature on the
ratio of bound active versus diluent strands can be explained
based on the model describing how DNA strands interact with
AuNPs during ligand exchange. To maintain nanoparticle
stability, DNA (unthiolated, disulfide-terminated, or thiolated)
must be added prior to adding salt, suggesting that DNA
strands adsorb rapidly and nonspecifically (via DNA bases) to
the AuNPs, preventing their aggregation.26,35,41,48
After adding
salt, nonthiolated DNA strands form sparse monolayers on
AuNPs, whereas thiolated DNA strands rearrange to permit
additional binding and form dense monolayers.43
This suggests
that nonthiolated DNA strands maintain a horizontal
orientation with respect to the AuNP surface, whereas thiolated
DNA strands initially adsorb in a horizontal orientation, then
adopt a vertical orientation after specific binding.43
When DNA−NPs are prepared by incubating disulfide-
terminated DNA strands with AuNPs at pH 3, an initial sparse
monolayer is rapidly attained,35
and our results suggest that the
added NaCl allows the bound DNA strands to rearrange and
form thiol bonds in an orientation that permits additional DNA
adsorption. At pH 3, the adenosine residues are protonated,49
thereby reducing their binding affinity for the gold. Adding salt
further reduces electrostatic repulsion between DNA strands on
the AuNPs and DNA in solution.26
DNA can thus rearrange
and additional binding can occur after adding the buffer and salt
to the ligand exchange reaction mixtures. During this
rearrangement and additional binding step, adsorption of the
bulky DNA1 ligand is hindered, resulting in an increase in the
DNA2 content on the surface of the AuNPs even when it is a
minor component of the ligand exchange mixture. This effect is
less for AuNPs with a larger radius of curvature, because the
gold surface is more accessible for binding.
The fact that the radius of curvature influenced the assembly
of DNA strands on the AuNPs therefore leads us to conclude
that disulfide-terminated DNA strands nonspecifically adsorb to
the AuNPs and rearrange to form specific bonds with the
AuNPs, following an adsorption mechanism similar to the two-
step model followed by thiolated DNA, rather than the one-
step adsorption model followed by nonthiolated DNA.43
■ CONCLUSION
We developed a rapid, convenient, and inexpensive method to
quantify the number of label-free DNA strands attached to
AuNPs of large or small core sizes. The number of strands per
nanoparticle can easily be determined from solutions of DNA−
NPs at concentrations typically used in sensing assays.
The UV−vis spectroscopy assay was used in concert with a
conventional Oligreen dye assay to determine two different
DNA sequences bound to AuNPs, without the need for labeled
DNA. The results of our mixed ligand shell analysis support a
model for disulfide-terminated DNA adsorption in which there
is fast nonspecific adsorption of DNA to the gold surface
dictated by chain length and base composition, followed by
rearrangement and additional specific binding to the gold
surface.
The generality of our approach means that, in principle, this
method can be extended to determine the number of DNA,
complementary DNA, RNA, or synthetic peptide strands
(whose UV−vis signatures overlap with that of decomposed
gold nanoparticles)50−52
bound to gold or silver nanoparticles.
These materials are of interest due to their ability to form
versatile nanoparticle assemblies,53,54
specifically induce
apoptosis in tumor cells,55
and act as sensitive analytical probes
in single-molecule experiments.54
The concentration of silver
nanoparticles can be determined from their UV−vis Aλmax and
empirically determined extinction coefficients.56
Solutions of
silver nanoparticles, at the concentration used for in vivo
toxicity assays,57
undergo oxidative decomposition by KCN58
to form salts that absorb light at 260 nm.38
Using this method
in concert with a dye that specifically binds double-stranded
Figure 6. (a) Calibration curves for determining A260 of decomposed
citrate-stabilized NPs. UV−vis spectroscopy can determine the DNA
bound to AuNPs in solutions containing ≥30 nM 5 nm AuNPs. The
linear fit for the decomposed 5 nm AuNPs had an R2
value of 0.990.
(b) Percentage DNA1 in ligand shell of 5 nm AuNPs functionalized
from different feed ratios of DNA1/DNA2 sequences. Error bars
represent the standard deviation for the mean % DNA1 in ligands
bound to 5 nm AuNPs.
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8. DNA would allow the number of bound complementary DNA
strands to be determined.
The simplicity and wide applicability of this method makes it
well-suited for determining the number of recognition and
diluent DNA strands bound to gold nanoparticles. This
information is essential to understanding the relationship
between the structure of a nanoparticle’s ligand shell and its
analytical and biosensing properties. We anticipate that
information gained using this method will lead to design of
nanomaterials with enhanced properties.
■ ASSOCIATED CONTENT
*S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.anal-
chem.6b02640.
TEM and SAXS characterization of AuNPs, fluorescent
spectroscopy analysis of DNA−NP purification, UV−vis
absorbance of commonly used DNA−NP purification
buffers, and fluorescent emission of mixtures of DNA
sequences and Oligreen dye (PDF)
■ AUTHOR INFORMATION
Corresponding Author
*Phone: 541-346-4228. E-mail: hutch@uoregon.edu.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
We thank the Center for Advanced Materials Characterization
in Oregon and the Institute of Molecular Biology for use of
their facilities and technical support. We thank the Materials
Science Institute and the University of Oregon Department of
Chemistry and Biochemistry for financial support. We thank
Andy Berglund for helpful discussions. We acknowledge the Air
Force Research Laboratory (under agreement FA 8650-05-1-
5041) for financial support.
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