Integrated nanostructures for direct detection of DNA at attomolar concentrations

Shih

2014

Analyst

We have examined real-time, in situ hybridization detection of target DNA (tDNA) in a buffer solution and in urine using 8 μm-thick lead magnesium niobate–lead titanate (PMN–PT) piezoelectric plate sensors (PEPSs) about 1.1–1.2 mm long and 0.45 mm wide with improved 3-mercaptopropyltrimethoxysilane (MPS) insulation and a new multiple-parabola (>50) resonance peak position fitting algorithm. With probe DNA (pDNA) immobilized on the PEPS surface and by monitoring the first width extension mode (WEM) resonance frequency shift we detected tDNA in real time at concentration as low as 1 × 10−19 M in urine (100 zM) with a signal to noise ratio (SNR) of 13 without DNA isolation and amplification at room temperature in 30 min. The present multiple-parabola fitting algorithm increased the detection of SNR by about 10 times compared to those obtained using the raw data and by about 5 times compared to those obtained using single parabola fitting. The detection was validated by in situ follow-up detection and subsequent visualization of fluorescent reporter microspheres (FRMs) coated with reporter DNA complementary to the tDNA but different from the probe pDNA.

Section: Introductionmentioning

confidence: 99%

DNA hybridization detection with 100 zM sensitivity using piezoelectric plate sensors with an improved noise-reduction algorithm

Shih

2014

Analyst

“…Peptide nucleic acid (PNA) probe-enhanced electrochemical biosensors based on an integrated chip also exhibited aM sensitivity. However, they also required washing 32 . Recently a disposable electrochemical biosensor based on magnetic bead amplification and target DNA biotinylation exhibited aM sensitivity 33 .…”

Section: Introductionmentioning

confidence: 99%

In situ, amplification-free double-stranded mutation detection at 60 copies/ml with thousand-fold wild type in urine

Biosensors and Bioelectronics

Lin

et al. 2018

We have investigated amplification-free in situ double-stranded mutation detection in urine in the concentration range 10 M - 10 M using piezoelectric plate sensors (PEPs). The detection was carried out in a close-loop flow with two temperature zones. The 95 °C high-temperature zone served as the reservoir where the sample was loaded and DNA de-hybridized. The heated urine was cooled flowing through a 1 m long tubing immersed in room-temperature water bath at a flow rate of 4 ml/min to reach the detection cell at the desired temperature for the detection to take place. With hepatitis B virus double mutation (HBVDM) and KRAS G12V point mutation as model double mutations, it is shown that PEPS was able to detect double-stranded HBVDM and KRAS with 70% detection efficiency or better at concentration as low as 10 M against single-stranded mutation detection at the same concentrations, which was validated by the following in situ fluorescent reporter microspheres (FRMs) detection as well as microscopic visualization of the FRMs bound to the captured mutant on the PEPS surface. Furthermore, the same double-stranded mutation detection efficacy was demonstrated at 10 M - 10 M in a background of 250-fold wildtype for HBVDM and 1000-fold wildtype for KRAS. Also demonstrated was detection of KRAS mutation at 10 M - 10 M of SW480 DNA fragments in urine.

“…Peptide nucleic acid (PNA) probe-enhanced electrochemical biosensors that were based on an integrated chip also exhibited aM sensitivity. However, they also required washing 32 . Recently a disposable electrochemical biosensor based on magnetic bead amplification and target DNA biotinylation exhibited aM sensitivity 33 .…”

Section: Introductionmentioning

confidence: 99%

Amplification-free in situ KRAS point mutation detection at 60 copies per mL in urine in a background of 1000-fold wild type

Shih

2016

Analyst

We have examined in situ detection of single-nucleotide KRAS mutation in urine using a (Pb(Mg1/3Nb2/3)O3)0.65(PbTiO3)0.35 (PMN-PT) piezoelectric plate sensor (PEPS) coated with a 17-nucleotide (nt) locked nucleic acid (LNA) probe DNA complementary to the KRAS mutation. To enhance in situ mutant (MT) DNA detection specificity against the wild type (WT), the detection was carried out in a flow with a flow rate of 4 mL/min and at 63°C with the PEPS vertically situated at the center of the flow in which both the temperature and the flow impingement force discriminated the wild type. Under such conditions, PEPS was shown to specifically detect KRAS MT in situ with 60 copies/mL analytical sensitivity in a background of clinically-relevant 1000-fold more WT in 30 min without DNA isolation, amplification, or labeling. For validation, the detection was followed with detection in a mixture of blue MT fluorescent reporter microspheres (FRMs) (MT FRMs) that bound to only the captured MT and orange WT FRMs that bound to only the captured WT. Microscopic examinations showed that the captured blue MT FRMs still outnumbered the orange WT FRMs by a factor of 4 to 1 even though WT was 1000-fold of MT in urine. Finally, multiplexed specific mutation detection was demonstrated using a 6-PEPS array each with a probe DNA targeting one of the 6 codon-12 KRAS mutations.