Development of wearable sensors for tailored patient wound care

In recent years a specialist interest has developed worldwide in advanced wound management for difficult to heal chronic wounds. Further progress in advanced wound management will require an improvement in personalized medicine for the patient and in particular an improvement in the availability of diagnostic tests and parameters that fulfil clinical need in wound management decisions. However, without easy to use sensors for nurses and carers these potentially important near-patient diagnostic parameters will not enter clinical diagnostics. This study focuses on a number of metrics for wound condition and wound healing: wound fluid pH, wound moisture, and wound matrix metalloproteinases (MMP) enzyme activity. To observe these important markers state of the art sensors have been developed that are based on inexpensive sensing technologies that can be integrated within wound dressings. These sensors will enable the wound healing markers to be studied and profiled in clinics which will further enhance the understanding of these markers and their relationship in the complex healing process involved in chronic wound healing

A wearable wound moisture sensor as an indicator for wound dressing change : an observational study of wound moisture and status

Wound moisture is known to be a key parameter to ensure optimum healing conditions in wound care. This study tests the moisture content of wounds in normal practice in order to observe the moisture condition of the wound at the point of dressing change. This study is also the first large-scale observational study that investigates wound moisture status at dressing change. The Wound Sense sensor is a commercially available moisture sensor which sits directly on the wound in order to find the moisture status of the wound without disturbing or removing the dressing. The results show that of the 588 dressing changes recorded, 44⋅9% were made when the moisture reading was in the optimum moisture zone. Of the 30 patients recruited for this study, 11 patients had an optimum moisture reading for at least 50% of the measurements before dressing change. These results suggest that a large number of unnecessary dressing changes are being made. This is a significant finding of the study as it suggests that the protocols currently followed can be modified to allow fewer dressing changes and less disturbance of the healing wound bed

A New Restriction Endonuclease-Based Method for Highly-Specific Detection of DNA Targets from Methicillin-Resistant Staphylococcus aureus

Abstract PCR multiplexing has proven to be challenging, and thus has provided limited means for pathogen genotyping. We developed a new approach for analysis of PCR amplicons based on restriction endonuclease digestion. The first stage of the restriction enzyme assay is hybridization of a target DNA to immobilized complementary oligonucleotide probes that carry a molecular marker, horseradish peroxidase (HRP). At the second stage, a target-specific restriction enzyme is added, cleaving the target-probe duplex at the corresponding restriction site and releasing the HRP marker into solution, where it is quantified colorimetrically. The assay was tested for detection of the methicillin-resistant Staphylococcus aureus (MRSA) pathogen, using the mecA gene as a target. Calibration curves indicated that the limit of detection for both target oligonucleotide and PCR amplicon was approximately 1 nM. Sequences of target oligonucleotides were altered to demonstrate that (i) any mutation of the restriction site reduced the signal to zero; (ii) double and triple point mutations of sequences flanking the restriction site reduced restriction to 50-80% of the positive control; and (iii) a minimum of a 16-bp target-probe dsDNA hybrid was required for significant cleavage. Further experiments showed that the assay could detect the mecA amplicon from an unpurified PCR mixture with detection limits similar to those with standard fluorescence-based qPCR. Furthermore, addition of a large excess of heterologous genomic DNA did not affect amplicon detection. Specificity of the assay is very high because it involves two biorecognition steps. The proposed assay is low-cost and can be completed in less than 1 hour. Thus, we have demonstrated an efficient new approach for pathogen detection and amplicon genotyping in conjunction with various end-point and qPCR applications. The restriction enzyme assay may also be used for parallel analysis of multiple different amplicons from the same unpurified mixture in broad-range PCR applications

A new restriction endonuclease-based method for highly-specific detection of DNA targets from methicillin-resistant Staphylococcus aureus.

PCR multiplexing has proven to be challenging, and thus has provided limited means for pathogen genotyping. We developed a new approach for analysis of PCR amplicons based on restriction endonuclease digestion. The first stage of the restriction enzyme assay is hybridization of a target DNA to immobilized complementary oligonucleotide probes that carry a molecular marker, horseradish peroxidase (HRP). At the second stage, a target-specific restriction enzyme is added, cleaving the target-probe duplex at the corresponding restriction site and releasing the HRP marker into solution, where it is quantified colorimetrically. The assay was tested for detection of the methicillin-resistant Staphylococcus aureus (MRSA) pathogen, using the mecA gene as a target. Calibration curves indicated that the limit of detection for both target oligonucleotide and PCR amplicon was approximately 1 nM. Sequences of target oligonucleotides were altered to demonstrate that (i) any mutation of the restriction site reduced the signal to zero; (ii) double and triple point mutations of sequences flanking the restriction site reduced restriction to 50-80% of the positive control; and (iii) a minimum of a 16-bp target-probe dsDNA hybrid was required for significant cleavage. Further experiments showed that the assay could detect the mecA amplicon from an unpurified PCR mixture with detection limits similar to those with standard fluorescence-based qPCR. Furthermore, addition of a large excess of heterologous genomic DNA did not affect amplicon detection. Specificity of the assay is very high because it involves two biorecognition steps. The proposed assay is low-cost and can be completed in less than 1 hour. Thus, we have demonstrated an efficient new approach for pathogen detection and amplicon genotyping in conjunction with various end-point and qPCR applications. The restriction enzyme assay may also be used for parallel analysis of multiple different amplicons from the same unpurified mixture in broad-range PCR applications

Calibration curves generated with either the purified 196<i>mecA</i> amplicon (diamonds) or the unpurified PCR mixture (containing the target amplicon) (squares).

<p>The logarithmic trendlines were calculated in Excel, and proved to be identical for the purified and non-purified amplicons.</p

Effects of restriction site positioning within the ds DNA hybrid, and non-complementary loop addition.

<p>The HRP signals (bars) are expressed as percentages of the fully cognate positive control (40-mer). Target-probe duplexes shown below the bars consist of (1) the probe attached to the streptavidin-modified surface with biotin (bottom) and conjugated to HRP (top), and (2) a target of variable length, non-complementary ends, and/or loops. The <i>Bgl</i>II restriction site is indicated with thick horizontal lines. Target designations are the following: 5′ (or 3′), corresponds to the 5′ (or 3′) ends of the full length positive control; C, control (fully cognate), L, loop (addition of 5 or 10 nucleotides); rs5′ (or rs3′), the end of restriction site to which 0, 3, or 5 (+0, +3, +5) complementary nucleotides were added. For rs3′+0, two targets were prepared that had different non-complementary sequences flanking the 3′-end of the restriction site (rs3′+0-A, rs3′+0-G). The target oligonucleotide sequences are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097826#pone-0097826-t001" target="_blank">Table 1</a>.</p

Detection of the non-purified amplicon <i>mecA</i> in the presence of a large excess of heterologous (mouse) genomic DNA.

<p>Circles and diamonds show replicate experiments performed using the amplicon-containing PCR mixture, closed and open for addition of 100 or 0 ng of mouse DNA, respectively. The triangles show the negative control supplemented with 100 ng of mouse DNA, specifically, dilutions of the whole PCR mixture that were not subjected to thermocycling (no amplicon formation as verified by gel electrophoresis).</p

Effect of point mutations introduced into the target sequence.

<p>(<b>A</b>) Single, double and triple mutations were introduced between the target center and the 3′ end corresponding to the surface-immobilized terminus of the target-probe duplex. (<b>B</b>) Mutations were introduced between the target center and the 5′ end corresponding to the end of the target-probe duplex that was free in solution. HRP signals (bars) are expressed as the percentages of the fully cognate positive control (dark grey bar 40, for 40-mer). Target-probe duplexes shown below the bars consist of (1) the probe attached to the streptavidin-modified surface with biotin (bottom) and conjugated to HRP (top), and (2) a 40-mer target with 1–3 mutations shown with black ovals. The <i>Bgl</i>II restriction site is indicated with thick horizontal lines. Targets are named with ‘rs’ for mutations introduced within the restriction site, otherwise the target name contains the replacement nucleotide (mostly G) and position within the sequence, starting from the 5′ target end. The rs19+24 contained two mutations at the ends of the restriction site. Target oligonucleotide sequences are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097826#pone-0097826-t001" target="_blank">Table 1</a>.</p

General schematic of the restriction enzyme assay.

<p>(<b>A</b>) Surface immobilization of HRP conjugated to an oligonucleotide probe specific for a target gene of interest. (<b>B</b>) The target DNA (an oligonucleotide or a denatured PCR amplicon) is hybridized to the immobilized probe. (<b>C</b>) Addition of a restriction enzyme (Rrec) that recognizes and cleaves the target-probe ds DNA hybrid, resulting in release of the HRP marker into the reaction solution. (<b>D</b>) The reaction solution is transferred into a new well and mixed with an HRP substrate for colorimetric detection. For each target DNA molecule one HRP molecule is released, resulting in a linear dependence of the signal on the target DNA concentration. (<b>E</b>) Detailed schematic of the double stranded target-probe DNA duplex, with the specific restriction site shown in purple. HRP, horseradish peroxidase; B, biotin; SA, streptavidin.</p

The oligonucleotide probe and targets used in the current study.

<p>Capital letters show sequences that are cognate between a target oligonucleotide and the probe, with the restriction site shown in bold.</p>1<p>The total length of a target sequence that is complementary to the 40-mer probe MCA-BG.</p>2<p>Tm was calculated for a target-probe hybrid in PBS (150 mM Na⁺).</p