Immobilization of Lambda Exonuclease onto Polymer Micropillar Arrays for the Solid-Phase Digestion of dsDNAs

Oliver-Calixte, Nyoté J.; Uba, Franklin I.; Battle, Katherine E.; Weerakoon-Ratnayake, Kumuditha M.; Soper, Steven A.

doi:10.1021/ac5002965

Cited by 20 publications

(35 citation statements)

References 63 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In Figure a, the current reached a saturated value at high concentration of the hsDNA substrate, showing the characteristics of the Michaelis‐Menten kinetics. The K’ M determined in the study (Figure b) was calculated to be 90 nM which was close to the reported value for Exo III in homogeneous assay . These results revealed that there was no substantial loss of Exo III activity, indicating the method using electrochemical graphene utilization was comparable with other conventional approaches.…”

Section: Resultsmentioning

confidence: 99%

Homogeneous Electrochemical Assay for Real‐time Monitoring of Exonuclease III Activity Based on a Graphene Monolayer

et al. 2017

View full text Add to dashboard Cite

A homogeneous electrochemical assay based on a graphene monolayer electrode was developed for simple, sensitive, rapid and quantitative analysis of the exonuclease III (Exo III) activity. The method utilized a methylene blue (MB) tagged DNA substrate with hairpin structure, and a graphene monolayer attached on the working electrode. Before digestion, the hairpin structure prevents the adsorption of the DNA substrate to the graphene surface. Degradation of the substrate by the 3′–5′ Exo III, however, yields single‐stranded DNA (ssDNA), resulting in its subsequent binding to the graphene surface through π‐π stacking, which produces the voltammetric current from electrochemical reduction of the MB tag anchoring at the end of ssDNA. A direct quantification of the Exo III activity can be achieved by measuring the reductive peak current of the MB tag under easily attainable potential (∼ −0.1 V vs Ag/AgCl) range comparably sensitive to the conventional methods such as a gel‐based or fluorescence‐based assays. Our approach can be applied to measure various exonucleases activity by adjusting the structure of DNA substrate suggesting a new assay method in drug screening and basic research related to the enzymes.

show abstract

Section: Resultsmentioning

confidence: 99%

Homogeneous Electrochemical Assay for Real‐time Monitoring of Exonuclease III Activity Based on a Graphene Monolayer

et al. 2017

View full text Add to dashboard Cite

show abstract

“…Only O 2 plasma activation was explored here because this is commonly used for the assembly of nanofluidic devices, and surface roughness can play a significant role in determining the functional properties of nanofluidic devices. 9, 12, 17, 25, 41, 46, 47 For these studies, the initial RMS surface roughness of native COC thermoplastics was measured using AFM. Then, each substrate was exposed to 30 s of 50 W O 2 plasma, as this treatment can serve as an effective strategy for thermal fusion bonding cover plates to thermoplastic substrates, 46 and the RMS roughness was re-measured following this treatment.…”

Section: Resultsmentioning

confidence: 99%

Characterization of activated cyclic olefin copolymer: effects of ethylene/norbornene content on the physiochemical properties

O'Neil

Taylor

Ratnayake

et al. 2016

Analyst

Self Cite

View full text Add to dashboard Cite

The ethylene/norbornene content within cyclic olefin copolymer (COC) is well known to affect the chemical and physical properties of the copolymer, such as the glass transition temperature (Tg) and transparency. However, no work has been reported evaluating the effects of the ethylene/norbornene content on the surface properties of COC following UV/O3 or O2 plasma activation. Activation with either O2 plasma or UV/O3 is often used to assist in thermal assembly of fluidic devices, increasing the wettability of the surfaces, or generating functional scaffolds for the attachment of biological elements. Thus, we investigated differences in the physiochemical surface properties of various ethylene/norbornene compositions of COC following activation using analytical techniques such as water contact angle (WCA), ATR-FTIR, XPS, TOF-SIMS, UV-VIS, AFM and a colorimetric assay utilizing Toluidine Blue O (TBO). Results showed that increased norbornene content led to the generation of more oxygen containing functionalities such as alcohols, ketones, aldehydes and carboxyl groups when activated with either UV/O3 or O2 plasma. Specifically, COC with ~60% norbornene content showed a significantly higher –COOH functional group density when compared to COC with a 50% norbornene content and COC with a 35% norbornene content following UV/O3 or O2 plasma activation. Furthermore, COC with large norbornene contents showed a smaller average RMS roughness (0.65 nm) when compared to COC containing low norbornene contents (0.95 nm) following activation making this substrate especially suited for nanofluidic applications, which require smooth surfaces to minimize effects arising from dielectrophoretic trapping or non-specific adsorption. Although all COC substrates showed >90% transparency at wavelengths >475 nm, COC possessing high norbornene contents showed significantly less transparency at wavelengths below 475 nm following activation, making optical detection in this region difficult. Our data showed distinct physiochemical differences in activated COC that was dependent upon the ethylene/norbornene content of the thermoplastic and thus, careful selection of the particular COC grade must be considered for micro- and nanofluidics.

show abstract

“…To address these issues, we are developing an innovative SMS strategy that enzymatically cleaves DNAs using a surface-immobilized processive exonuclease to generate the dNMPs [24–26]. The released dNMPs are electrokinetically transported through a nanochannel and identification of the nucleobases is carried out using the unique apparent mobility ( i.e ., mobility matching) of each dNMP electrokinetically traveling through a nanochannel.…”

Section: Introductionmentioning

confidence: 99%

Electrokinetic transport properties of deoxynucleotide monophosphates (dNMPs) through thermoplastic nanochannels

O'Neil

Amarasekara

Weerakoon-Ratnayake

et al. 2018

Analytica Chimica Acta

Self Cite

View full text Add to dashboard Cite

The electrokinetic behavior of molecules in nanochannels (<100 nm in length) have generated interest due to the unique transport properties observed that are not seen in microscale channels. These nanoscale dependent transport properties include transverse electromigration arising from partial electrical double layer overlap, enhanced solute/wall interactions due to the small channel diameter, and field-dependent intermittent motion produced by surface roughness. In this study, the electrokinetic transport properties of deoxynucleotide monophosphates (dNMPs) were investigated, including the effects of electric field strength, surface effects, and composition of the carrier electrolyte (ionic concentration and pH). The dNMPs were labeled with a fluorescent reporter (ATTO 532) to allow tracking of the electrokinetic transport of the dNMPs through a thermoplastic nanochannel fabricated via nanoimprinting (110 nm × 110 nm, width × depth, and 100 μm in length). We discovered that the transport properties in plastic nanochannels of the dye-labeled dNMPs produced differences in their apparent mobilities that were not seen using microscale columns. We built histograms for each dNMP from their apparent mobilities under different operating conditions and fit the histograms to Gaussian functions from which the separation resolution could be deduced as a metric to gage the ability to identify the molecule based on their apparent mobility. We found that the resolution ranged from 0.73 to 2.13 at pH = 8.3. Changing the carrier electrolyte pH > 10 significantly improved separation resolution (0.80-4.84) and reduced the standard deviation in the Gaussian fit to the apparent mobilities. At low buffer concentrations, decreases in separation resolution and increased standard deviations in Gaussian fits to the apparent mobilities of dNMPs were observed due to the increased thickness of the electric double layer leading to a partial parabolic flow profile. The results secured for the dNMPs in thermoplastic nanochannels revealed a high identification efficiency (>99%) in most cases for the dNMPs due to differences in their apparent mobilities when using nanochannels, which could not be achieved using microscale columns.

show abstract

Immobilization of Lambda Exonuclease onto Polymer Micropillar Arrays for the Solid-Phase Digestion of dsDNAs

Cited by 20 publications

References 63 publications

Homogeneous Electrochemical Assay for Real‐time Monitoring of Exonuclease III Activity Based on a Graphene Monolayer

Homogeneous Electrochemical Assay for Real‐time Monitoring of Exonuclease III Activity Based on a Graphene Monolayer

Characterization of activated cyclic olefin copolymer: effects of ethylene/norbornene content on the physiochemical properties

Electrokinetic transport properties of deoxynucleotide monophosphates (dNMPs) through thermoplastic nanochannels

Contact Info

Product

Resources

About