New Detection Modality for Label-Free Quantification of DNA in Biological Samples via Superparamagnetic Bead Aggregation

Leslie, Daniel C.; Li, Jingyi; Strachan, Briony C.; Begley, Matthew R.; Finkler, David; Bazydlo, Lindsay A.L.; Barker, Nathaniel; Haverstick, Doris M.; Utz, Marcel; Landers, James P.

doi:10.1021/ja300839n

Cited by 52 publications

(84 citation statements)

References 34 publications

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“…By using the assumption that each WBC contains approximately 6 pg DNA (9), the aggregation caused by DNA released from lysed WBCs can be correlated to the cell count provided by the clinical instruments at the UVA Clinical Laboratory, after back calculation (5). Based on the analysis of a series of WBC sample dilution standard curves, a sample dilution factor of 3200× yielded results with the largest % dispersion dynamic range (72% to 34% dispersion) across the range of WBC counts observed in patient samples and showed the smallest amount of variability between samples in terms of correlation value (R 2 = 0.955) compared to other dilution factors between 1600× and 12 800×.…”

Section: Focused Reportsmentioning

confidence: 99%

Use of Dual-Force Aggregation as a Multiplexed, Rapid Point-of-Care Screening Method for White Blood Cell Counting from Whole Blood Samples

Nelson

Hughes

Engel

et al. 2017

The Journal of Applied Laboratory Medicine

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Background Enumeration of blood cells is an integral metric for evaluating patient health and can be used to screen for a wide range of diseases and conditions. Conventional methods rely on large, expensive, and complicated instrumentation that requires trained technicians and is not amenable to point-of-care analysis. This work demonstrates the use of a multiplexed, bead-based assay for both rapid white blood cell (WBC) count screening and accurate, multiplexed WBC counts for point-of-care analysis. Methods Blood samples were lysed and diluted before being incubated with silica-coated magnetic particles under chaotropic conditions, a rotating magnetic field, and a source of agitation. The resulting bead aggregation was imaged and correlated to a known WBC count. After establishing standard curves, the WBC count for 18 whole blood samples were determined by this method and compared to values obtained conventionally. Results When the optimal dilution factor for lysis of whole blood samples was established, 17 of 18 samples (94.4%) were correctly screened and categorized as having high, typical, or low WBC count, while 14 of 18 samples were within 16% of the reported clinical values. The developed system provides analysis of 13 samples in <3 min with a total analysis time of approximately 10 min (including incubation and dilution) and represents comparable throughput to conventional instrumentation, while providing point-of-care capability with reduced size (14 × 21 × 14 cm) and simplicity. Conclusions This work demonstrates the potential for a multiplexed, bead-based assay to be used as a rapid, point-of-care screening method for WBC counting from whole blood samples.

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Section: Focused Reportsmentioning

confidence: 99%

Use of Dual-Force Aggregation as a Multiplexed, Rapid Point-of-Care Screening Method for White Blood Cell Counting from Whole Blood Samples

Nelson

Hughes

Engel

et al. 2017

The Journal of Applied Laboratory Medicine

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“…24,25 HIA utilizes a pair of oligonucleotide probes that are complementary to the target DNA sequence. The probes are immobilized on the surface of paramagnetic particles ( via biotin-streptavidin conjugation), and their interaction with target DNA is enhanced by a rotating magnetic field (RMF).…”

Section: Introductionmentioning

confidence: 99%

A simple integrated microfluidic device for the multiplexed fluorescence-free detection of Salmonella enterica

Strachan

Sloane

Houpt

et al. 2016

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Rapid, inexpensive and simplistic nucleic acid testing (NAT) is pivotal in delivering biotechnology solutions at the point-of-care (POC). We present a poly(methylmethacrylate) (PMMA) microdevice where on-board infrared-mediated PCR amplification is seamlessly integrated with a particle-based, visual DNA detection for specific detection of bacterial targets in less than 35 minutes. Fluidic control is achieved using a capillary burst valve laser-ablated in a novel manner to confine the PCR reagents to a chamber during thermal cycling, and a manual torque-actuated pressure system to mobilize the fluid from the PCR chamber to the detection reservoir containing oligonucleotide-adducted magnetic particles. Interaction of amplified products specific to the target organism with the beads in a rotating magnetic field allows for near instantaneous (<30 s) detection based on hybridization-induced aggregation (HIA) of the particles and simple optical analysis. The integration of PCR with this rapid, sequence-specific DNA detection method on a single microdevice presents the possibility of creating POC NAT systems that are low cost, easy-to-use, and involve minimal external hardware.

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“…22 The method ( Figure 1) is an extension of hybridization-induced aggregation (HIA) technology, whereby the hybridization of a specific DNA target to a pair of oligonucleotide probes immobilized on the surface of microbeads, tethers the microbeads together and induces aggregation. 23,24 A digital image of the hybridization microwell is used for quantifying the extent of aggregation in terms of image saturation, which allows for a quantitative representation of hybridization efficiency. We exploit this phenomenon to differentiate target DNA sequences that differ by only a single nucleotide in a method we refer to as HIA for mutation detection (HIA MD ).…”

mentioning

confidence: 99%

Hybridization-Induced Aggregation Technology for Practical Clinical Testing

Sloane

Landers

Kelly

2016

The Journal of Molecular Diagnostics

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KRAS mutations have emerged as powerful predictors of response to targeted therapies in the treatment of lung and colorectal cancers; thus, prospective KRAS genotyping is essential for appropriate treatment stratification. Conventional mutation testing technologies are not ideal for routine clinical screening, as they often involve complex, time-consuming processes and/or costly instrumentation. In response, we recently introduced a unique analytical strategy for revealing KRAS mutations, based on the allele-specific hybridization-induced aggregation (HIA) of oligonucleotide probe-conjugated microbeads. Using simple, inexpensive instrumentation, this approach allows for the detection of any common KRAS mutation in <10 minutes after PCR. Here, we evaluate the clinical utility of the HIA method for mutation detection (HIAMD). In the analysis of 20 lung and colon tumor pathology specimens, we observed a 100% correlation between the KRAS mutation statuses determined by HIAMD and sequencing. In addition, we were able to detect KRAS mutations in a background of 75% wild-type DNA-a finding consistent with that reported for sequencing. With this, we show that HIAMD allows for the rapid and cost-effective detection of KRAS mutations, without compromising analytical performance. These results indicate the validity of HIAMD as a mutation-testing technology suitable for practical clinical testing. Further expansion of this platform may involve the detection of mutations in other key oncogenic pathways.

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New Detection Modality for Label-Free Quantification of DNA in Biological Samples via Superparamagnetic Bead Aggregation

Cited by 52 publications

References 34 publications

Use of Dual-Force Aggregation as a Multiplexed, Rapid Point-of-Care Screening Method for White Blood Cell Counting from Whole Blood Samples

Use of Dual-Force Aggregation as a Multiplexed, Rapid Point-of-Care Screening Method for White Blood Cell Counting from Whole Blood Samples

A simple integrated microfluidic device for the multiplexed fluorescence-free detection of Salmonella enterica

Hybridization-Induced Aggregation Technology for Practical Clinical Testing

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