Parallel nanoliter detection of cancer markers using polymer microchips

Gulliksen, Anja; Solli, Lars; Drese, Klaus; Sörensen, O.; Karlsen, Frank; Rogne, Henrik; Hovig, Eivind; Sirevåg, Reidun

doi:10.1039/b415525d

Cited by 87 publications

(63 citation statements)

References 14 publications

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“…This technology has already been adapted for performing hands-free Nucleic Acid Diagnostic (NAD) (Gulliksen et al, 2005), including complete extraction of RNA and DNA (Baier et al, 2009). The disposable chips are designed to include all reagents needed for complete NAD, including the detection of different mRNA targets, and may be mass produced to a low cost adapted to a low-resource market.…”

Section: Discussionmentioning

confidence: 99%

A comprehensive evaluation of the accuracy of cervical pre-cancer detection methods in a high-risk area in East Congo

et al. 2010

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BACKGROUND: Given the high burden of cervical cancer in low-income settings, there is a need for a convenient and affordable method for detecting and treating pre-cancerous lesions. METHODS: Samples for comparing the accuracy of cytology, virology and histology were collected. Identification of HPV E6/E7 mRNA was performed using PreTect HPV-Proofer. HPV DNA detection was performed by GP5 þ /6 þ PCR, followed by reverse line blot (RLB) for typing. RESULTS: A total of 343 women, aged 25 -60 years, attending gynaecological polyclinics in DR Congo were included for sample enrolment. The test positivity rate was conventional and liquid-based cytology (LBC) at cutoff ASCUS þ of 6.9 and 6.6%, respectively; PreTect HPV-Proofer of 7.3%; and consensus DNA PCR for 14 HR types of 18.5%. Sixteen cases of CIN2 þ lesions were identified. Of these, conventional cytology identified 66.7% with a specificity of 96.2%, LBC identified 73.3% with a specificity of 96.9%, all at cutoff ASCUS þ. HR-HPV DNA detected all CIN2 þ cases with a specificity of 85.9%, whereas PreTect HPV-Proofer gave a sensitivity of 81.3% and a specificity of 96.6%. CONCLUSION: Both HPV detection assays showed a higher sensitivity for CIN2 þ than did cytological methods. Detecting E6/E7 mRNA from only a subset of HR HPVs, as is the case with PreTect HPV-Proofer, resulted in a similar specificity to cytology and a significantly higher specificity than consensus HR HPV DNA (Po0.0001).

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Section: Discussionmentioning

confidence: 99%

A comprehensive evaluation of the accuracy of cervical pre-cancer detection methods in a high-risk area in East Congo

et al. 2010

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“…The first integration of a NASBA reaction into a microfluidic silicon chip was shown in 2005 by Gulliksen et al [83]. They have used the molecular beacon strategy to show sensitivities comparable to laboratory based NASBA reactions.…”

Section: Nasba: Nucleic Acid Sequence-based Amplificationmentioning

confidence: 99%

Isothermal Amplification and Quantification of Nucleic Acids and its Use in Microsystems

Tröger¹,

Niemann²,

Gärtig³

et al. 2015

J Nanomed Nanotechnol

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Nucleic acid amplification technologies (NAATs) offer the most sensitive tests in the clinical laboratory. These techniques are used as a powerful tool for screening and diagnosis of infectious diseases. Isothermal methods, as an alternative to polymerase chain reaction (PCR), require no thermocycling machine and can mostly be performed with reduced time, high throughput, and accurate and reliable results.However, current molecular diagnostic approaches generally need manual analysis by qualified and experienced personal which is a highly complex, time-consuming and labor-intensive task. Thus, the demand for simpler, miniaturized systems and assays for pathogen detection is steadily increasing. Microfluidic platforms and lab-on-a-chip devices have many advantages such as small sample volume, portability and rapid detection time and enable point-of-care diagnosis.In this article, we review several isothermal amplification methods and their implementation in microsystems in relation to quantification of nucleic acids. miniaturized systems can be diverse. The majority of publications are focussed on clinical diagnostics including foodborne organisms for environmental monitoring [10][11][12]. In case of an infectious disease, cultivation and phenotypic characterization are still the standard methods of microbiologists which need days up to weeks to obtain a diagnostic result. Hence, scientists started to make use of nucleic acid amplification methods to accelerate the analysis. The most widespread and well known technique for this purpose is the polymerase chain reaction (PCR) [10,13], which exploits the activity of the DNA polymerase. The method is based on a thermal cycling process consisting of repeated cycles of heating and cooling steps allowing for DNA melting, sequence specific primer binding and enzymatic amplification of the target nucleic acid. The quantitative assessment of target copies is possible by using a real-time PCR approach, where a concentration dependent signal (e.g. fluorescence) increases over time [14]. The latter enables the user to easily assess the pathogen load of patient samples [15][16][17][18]. Since the first example of a PCR on a miniaturized system in 1994 [19,20], numerous devices were presented, which integrate not just the amplification, but also sample preparation and detection steps [9,[21][22][23][24][25][26][27][28]. Just a few of those microsystems have reached the market so far, but some have shown a fascinating potential to replace the classical laboratory-oriented state-of-the art [29][30][31][32][33]. setups has been shown to obtain a similar result [34]. A smaller heat capacity allows for rapid changes in temperature beneficial for the PCR time as well as a higher parallelism of multiple genetic samples [34].However, a major drawback of the integration of PCR to microsystems is the necessity of sophisticated instrumentation. The reaction requires a thermal cycling instrumentation, space and considerable expertise [35]. Therefore, isothermal reactions for the ta...

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“…They demonstrated an improvement to the first design in 2005, 30 showing that microfluidic real-time NASBA performed as well as conventional off-chip (in-tube) real-time NASBA.…”

Section: 19mentioning

confidence: 99%

Integrated microfluidic tmRNA purification and real-time NASBA device for molecular diagnostics

et al. 2008

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We demonstrate the first integrated microfluidic tmRNA purification and nucleic acid sequence-based amplification (NASBA) device incorporating real-time detection. The real-time amplification and detection step produces pathogen-specific response in < 3 min from the chip-purified RNA from 100 lysed bacteria. On-chip RNA purification uses a new silica bead immobilization method. On-chip amplification uses custom-designed high-selectivity primers and real-time detection uses molecular beacon fluorescent probe technology; both are integrated on-chip with NASBA. Present in all bacteria, tmRNA (10Sa RNA) includes organism-specific identification sequences, exhibits unusually high stability relative to mRNA, and has high copy number per organism; the latter two factors improve the limit of detection, accelerate time-to-positive response, and suit this approach ideally to the detection of small numbers of bacteria. Device efficacy was demonstrated by integrated on-chip purification, amplification, and real-time detection of 100 E. coli bacteria in 100 mL of crude lysate in under 30 min for the entire process.

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Parallel nanoliter detection of cancer markers using polymer microchips

Cited by 87 publications

References 14 publications

A comprehensive evaluation of the accuracy of cervical pre-cancer detection methods in a high-risk area in East Congo

A comprehensive evaluation of the accuracy of cervical pre-cancer detection methods in a high-risk area in East Congo

Isothermal Amplification and Quantification of Nucleic Acids and its Use in Microsystems

Integrated microfluidic tmRNA purification and real-time NASBA device for molecular diagnostics

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