A microfluidic platform for transcription- and amplification-free detection of zepto-mole amounts of nucleic acid molecules

Mayr, Reinhard; Haider, Michaela; Thünauer, Roland; Haselgrübler, Thomas; Schütz, Gerhard J.; Sonnleitner, Alois; Hesse, Jan

doi:10.1016/j.bios.2015.11.013

Cited by 21 publications

(18 citation statements)

References 30 publications

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“…Fluorescent labeling of target mRNA was achieved by hybridizing 5′‑Cyanine 5 (Cy5)‑labeled complementary DNA probes (subsequently denoted as labels) to a different section of the mRNA. This approach has been shown before for the ultra-sensitive detection of in vitro synthesized short DNA and RNA molecules in a dynamic microfluidic chip [22]. …”

Section: Methodsmentioning

confidence: 99%

“…Analysis of the average spot brightness was performed with MATLAB (MathWorks TM Inc., Natick, MA, USA) as previously described [22]. In brief, for each spot, a sub-image was extracted from the raw data for further processing.…”

Section: Methodsmentioning

confidence: 99%

“…Our group has recently shown an enzyme-free double-hybridization assay for the specific detection of short DNA and RNA oligonucleotides on a microarray with single-molecule sensitivity [22]. In the present report, we optimized this approach to enable the analysis of endogenous mRNA of a housekeeping gene contained in isolated total RNA.…”

Section: Introductionmentioning

confidence: 99%

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A Double-Hybridization Approach for the Transcription- and Amplification-Free Detection of Specific mRNA on a Microarray

Haider¹,

Haselgrübler²,

Sonnleitner³

et al. 2016

Microarrays

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A double-hybridization approach was developed for the enzyme-free detection of specific mRNA of a housekeeping gene. Targeted mRNA was immobilized by hybridization to complementary DNA capture probes spotted onto a microarray. A second hybridization step of Cy5-conjugated label DNA to another section of the mRNA enabled specific labeling of the target. Thus, enzymatic artifacts could be avoided by omitting transcription and amplification steps. This manuscript describes the development of capture probe molecules used in the transcription- and amplification-free analysis of RPLP0 mRNA in isolated total RNA. An increase in specific signal was found with increasing length of the target-specific section of capture probes. Unspecific signal comprising spot autofluorescence and unspecific label binding did not correlate with the capture length. An additional spacer between the specific part of the capture probe and the substrate attachment site increased the signal significantly only on a short capture probe of approximately 30 nt length.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

A Double-Hybridization Approach for the Transcription- and Amplification-Free Detection of Specific mRNA on a Microarray

Haider¹,

Haselgrübler²,

Sonnleitner³

et al. 2016

Microarrays

Self Cite

View full text Add to dashboard Cite

show abstract

“…In addition, the presence of inhibitors can prevent enzymatic amplification, thus target purification is often required (Bessetti, 2007). Finally, the inherent biases in enzymatic amplifications may prevent accurate quantification (Mayr et al, 2016). …”

Section: Introductionmentioning

confidence: 99%

Sensitive DNA detection and SNP discrimination using ultrabright SERS nanorattles and magnetic beads for malaria diagnostics

Ngo

Gandra

Fales

et al. 2016

Biosensors and Bioelectronics

118

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One of the major obstacles to implement nucleic acid-based molecular diagnostics at the point-of-care (POC) and in resource-limited settings is the lack of sensitive and practical DNA detection methods that can be seamlessly integrated into portable platforms. Herein we present a sensitive yet simple DNA detection method using a surface-enhanced Raman scattering (SERS) nanoplatform: the ultrabright SERS nanorattle. The method, referred to as the nanorattle-based method, involves sandwich hybridization of magnetic beads that are loaded with capture probes, target sequences, and ultrabright SERS nanorattles that are loaded with reporter probes. Upon hybridization, a magnet was applied to concentrate the hybridization sandwiches at a detection spot for SERS measurements. The ultrabright SERS nanorattles, composed of a core and a shell with resonance Raman reporters loaded in the gap space between the core and the shell, serve as SERS tags for signal detection. Using this method, a specific DNA sequence of the malaria parasite Plasmodium falciparum could be detected with a detection limit of approximately 100 attomoles. Single nucleotide polymorphism (SNP) discrimination of wild type malaria DNA and mutant malaria DNA, which confers resistance to artemisinin drugs, was also demonstrated. These test models demonstrate the molecular diagnostic potential of the nanorattle-based method to both detect and genotype infectious pathogens. Furthermore, the method’s simplicity makes it a suitable candidate for integration into portable platforms for POC and in resource-limited settings applications.

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“…[1][2][3] Various techniques have been developed for the sensitive detection of DNA, such as radiochemical, fluorescent, electrochemical, colorimetric, and chemiluminescent methods, etc. Electrochemical detection platforms have been widely applied, which benefit from a fast response, reliable, simple instrumentation, ease of miniaturization, high sensitivity and selectivity.…”

Section: Introductionmentioning

confidence: 99%

A Novel Electrochemical Sensor Based on [Ru(NH3)6]Cl3 as a Redox Indicator for the Detection of G-G Mismatched DNA

Zhu

Huang

et al. 2017

ANAL. SCI.

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Other mismatched DNA produced only a low response, as well as complementary DNA. Thus G-G mismatched DNA can be easily discriminated from other mismatched and complementary DNA based on the sensor. Furthermore, the method was simple, rapid and repeatable for the detection of G-G mismatched DNA. The selective detection of target dsDNA was achieved by a relative current ratio of the target and control DNA. These results demonstrated that this strategy could provide great promise for the rapid and specific detection of other sequence-specific DNA.

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A microfluidic platform for transcription- and amplification-free detection of zepto-mole amounts of nucleic acid molecules

Cited by 21 publications

References 30 publications

A Double-Hybridization Approach for the Transcription- and Amplification-Free Detection of Specific mRNA on a Microarray

A Double-Hybridization Approach for the Transcription- and Amplification-Free Detection of Specific mRNA on a Microarray

Sensitive DNA detection and SNP discrimination using ultrabright SERS nanorattles and magnetic beads for malaria diagnostics

A Novel Electrochemical Sensor Based on [Ru(NH3)6]Cl3 as a Redox Indicator for the Detection of G-G Mismatched DNA

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