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The highly enhanced binding properties of deoxyribonucleic acid (DNA)-gold nanoparticle conjugates (DNA-AuNPs) have attracted researchers' attention because they could be the fundamental origin of the high sensitivity of detection schemes employing the DNA-AuNPs as functional nanoprobes.1 -3 The binding constant of the DNA-AuNPs was determined to be of two orders of magnitude as high as that of free single stranded DNA, which was not the case with sparsely conjugated DNA strands on nanoparticles.4,5 In a typical conjugation procedure, citrate-coated AuNPs are combined with excess thiol DNA, which essentially leads to the limited DNA loading per AuNP and the discard of unconjugated costly thiol DNA (approximately 95%, assuming 100 DNA strands per AuNP).1 To date, several chemical and physical methods have been developed to effectively increase the DNA density on AuNPs. For example, DNAAuNP conjugation reaction was conducted at high NaCl concentrations, 6 with sonication, 6 or under acidic (pH 3) conditions 7 to increase the density of DNA on AuNPs. While effective and convenient, however, these methods are hampered by the necessity of additional chemicals or instrumentation.In contrast to the DNA-conjugation reaction, the detailed chemical conditions where the DNA strands are released from the nanoparticle surface have been less frequently investigated. In fact, the complete release of DNA from nanoparticles is as important as the DNA-nanoparticle conjugation, because it is directly associated with the accurate evaluation of DNA loading, a measure of the DNA conjugation efficiency. Conventionally, organic alkylthiol molecules in excess were employed to displace the monothiol DNA, but they were hampered by the poor water solubility and environmental harm.8 Instead of the displacing chemicals, potassium cyanide (KCN) was used to dissolve the gold nanoparticles, whose toxicity, however, could be an even larger concern than that of alkylthiols.9,10 Recently, sodium borohydride (NaBH 4 ) was used to release organic thiol molecules from AuNPs, potentially applicable to the DNA release.11 Currently, dithiothreitol (DTT) is the most widely used displacing reagent for thiol DNA on AuNPs because of its high water solubility, low toxicity, and facile removal after the displacement. 12 The detailed displacement conditions, however, are not fully investigated yet, and are required to be further improved for efficient and optimized quantification of DNA strands conjugated with AuNPs.Herein, we present a facile and cost-effective method for maximizing DNA loading on AuNPs simply by increasing the AuNP concentration and thus shifting chemical equilibrium of the DNA-AuNP conjugation reaction (Scheme 1(a)). Importantly, this method is extremely simple, straightforward, and can be applied to any DNA sequences. Moreover, it does not require any additional instrumentation or chemical reagents. For the accurate measurement of DNA loading, we first systematically investigated the DTT displacement of DNA strands on AuNPs at various tempe...
The highly enhanced binding properties of deoxyribonucleic acid (DNA)-gold nanoparticle conjugates (DNA-AuNPs) have attracted researchers' attention because they could be the fundamental origin of the high sensitivity of detection schemes employing the DNA-AuNPs as functional nanoprobes.1 -3 The binding constant of the DNA-AuNPs was determined to be of two orders of magnitude as high as that of free single stranded DNA, which was not the case with sparsely conjugated DNA strands on nanoparticles.4,5 In a typical conjugation procedure, citrate-coated AuNPs are combined with excess thiol DNA, which essentially leads to the limited DNA loading per AuNP and the discard of unconjugated costly thiol DNA (approximately 95%, assuming 100 DNA strands per AuNP).1 To date, several chemical and physical methods have been developed to effectively increase the DNA density on AuNPs. For example, DNAAuNP conjugation reaction was conducted at high NaCl concentrations, 6 with sonication, 6 or under acidic (pH 3) conditions 7 to increase the density of DNA on AuNPs. While effective and convenient, however, these methods are hampered by the necessity of additional chemicals or instrumentation.In contrast to the DNA-conjugation reaction, the detailed chemical conditions where the DNA strands are released from the nanoparticle surface have been less frequently investigated. In fact, the complete release of DNA from nanoparticles is as important as the DNA-nanoparticle conjugation, because it is directly associated with the accurate evaluation of DNA loading, a measure of the DNA conjugation efficiency. Conventionally, organic alkylthiol molecules in excess were employed to displace the monothiol DNA, but they were hampered by the poor water solubility and environmental harm.8 Instead of the displacing chemicals, potassium cyanide (KCN) was used to dissolve the gold nanoparticles, whose toxicity, however, could be an even larger concern than that of alkylthiols.9,10 Recently, sodium borohydride (NaBH 4 ) was used to release organic thiol molecules from AuNPs, potentially applicable to the DNA release.11 Currently, dithiothreitol (DTT) is the most widely used displacing reagent for thiol DNA on AuNPs because of its high water solubility, low toxicity, and facile removal after the displacement. 12 The detailed displacement conditions, however, are not fully investigated yet, and are required to be further improved for efficient and optimized quantification of DNA strands conjugated with AuNPs.Herein, we present a facile and cost-effective method for maximizing DNA loading on AuNPs simply by increasing the AuNP concentration and thus shifting chemical equilibrium of the DNA-AuNP conjugation reaction (Scheme 1(a)). Importantly, this method is extremely simple, straightforward, and can be applied to any DNA sequences. Moreover, it does not require any additional instrumentation or chemical reagents. For the accurate measurement of DNA loading, we first systematically investigated the DTT displacement of DNA strands on AuNPs at various tempe...
Herein, we designed and developed a simple, rapid and sensitive method for detection of coralyne and heparin using a singly labeled fluorescent probe, based on photoinduced electron transfer (PIET). A specific oligonucleotide strand was labeled with fluorophore (6‐FAM) at its 5’ end and four guanines at its 3’ end. In the presence of coralyne, homo‐adenine DNA duplex is formed via A2‐coralyne‐A2 coordination, thus leading to the fluorescence quenching due to PIET between guanine and fluorophore. Upon the addition of heparin, the formation of heparin‐coralyne complex can prevent coralyne from forming A2‐coralyne‐A2 coordination, separating fluorophore from guanines; as a result, the fluorescence intensity can be recovered. The detection limits of the method with respect to coralyne and heparin were 3.1 nM and 0.04 nM (S/N=3), respectively. Moreover, the proposed method was successfully applied to detect heparin in human serum sample, further demonstrating that it can have promising practical applications.
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