1.      Introduction

Human skin has a remarkable capacity to repair or regenerate upon wounding. However, occasionally if wound size exceeds certain limits or if the wound aetiology gives rise to it dedicated treatment by medical experts is required. In general, skin wounds are classified in acute and hard-to-heal wounds (Figure 1). Of the acute wound the burn wounds as well as hard to heal wounds not only have a tremendous impact on the quality of life of the patients, but also greatly affect healthcare systems due to high evoked costs[1]. Burn and hard-to-heal wounds often require intensive long-term treatment.

treatment starts to be needed[1]. However, this is generally connected with frequent and often painful replacement of the wound dressing. Every unnecessary wound dressing replacement, however, impedes the progress of the wound healing process. The problem of unnecessary wound care interventions was shown in a recent report, where around 85% of the wounds that underwent a change of wound dressings were actually not infected [2]. Knowing the wound condition beforehand such unnecessary dressing replacements could have been avoided[3]. Currently, control of the wound generally occurs by visual inspection after removal of the non-transparent wound dressing. One way is to solve this is making wound dressing fully transparent [4]. However, even with making visual inspection possible, it may be too late. If the latter visual inspection justifies additional tests, wound alterations are already in a rather late pathological state [5]. A prompt intervention at the earliest time point, at which normal healing changes towards a pathological process, may overcome a tedious and costly wound care management procedure. Therefore, it is of paramount importance for optimal wound therapy, to know how wound characteristics change over time. For this, however, smart wound dressings that allow non-invasive (semi-)continuous monitoring of these properties are necessary. While it is still a long road before such dressings will be available for routine clinical application, recent publications[6,7]and patents (e.g. WO2015168720, US2014298927 and US2014298928) indicate that we are on the verge of developing the next generation of wound dressings, which not only support wound healing but which are also “Smart” due to the presence of sensors monitoring the state of the wound. For this however, such biosensors need to fulfil a range of specifications including: (i) high specificity for the target (specific protein, sugar, lipid, ECM component, etc.), (ii) sensitivity in the relevant concentration-range, (iii) stability for real-time applications over a period of days to weeks, (iv) self-regenerating to enable multiple measurements of the target at varying concentrations and (v) modular organisation to allow assemblies of multiple sensors as 2D-array. The latter is of crucial importance for most target parameters since the wound state may diverge between the locations within the wound.

In the present review, after a short overview of wounds including bacterial contamination, current concepts for smart dressings that monitor wound state characteristics are described. Additionally, some gaps are identified that need to be filled before major progress in this field can be made. Furthermore, a foresight is given on what developments we may expect in future.

2.      Background

2.1.  Wounds

            2.1.1. Normal Wound Healing:The skin is a layered structure which can be subdivided in: (i) the non-vascularized epidermis as top layer mainly consisting of keratinocytes, (ii) the dermis as vascularized connective tissue mainly with fibroblasts, macrophages, hair follicles, glands, lymphatic vessels and nerve endings, and (iii) hypodermis containing mainly large blood vessels, fibroblasts and adipocytes. The skin represents an important barrier protecting the body for microbial attack and fluid loss. After wounding these structures are locally (partially) disrupted and repair processes start. Whereas superficial-layer wounds still have the hair follicle reservoir to heal, in case of full-thickness wounds can only heal from the edges.As a result, in the latter case local differences in wound chemistry are expected at different locations within the wound. Normal wound healing represents an orchestrated cascade of cellular and biochemical processes. It can be divided in at least 4 overlapping phases, i.e., haemostasis, inflammation, granulation (also called proliferation or fibroblastic phase) and remodelling.

At the initial phases at which wound is not closed by a scab wound fluid will be secreted. Due to the injured vasculature or vascular leakage wound fluid composition of acute surgical and traumatic wounds reflects at the beginning largely that of blood serum. However, during and after closure of vascular defects the wound fluid content changes and is suggested to contain among others high levels of Interleukin (IL)-6 and IL-8, as well as elevated levels of Monocyte Chemoattractant Protein (MCP)-1 (also termed C-C motif-chemokine ligand 2 or CCL2), IL-1 Receptor Antagonist(RA), Platelet Derived Growth Factor (PDGF), interferon gamma-Induced Protein (IP)-10 (also termed C-X-C motif chemokine 10 or CXCL10), Interferon(IFN)-γ and Tumour Necrosis Factor (TNF)-α with relatively low levels of IL-1β and IL-10 as measured 1 day after a surgical wounding [8]. After a first rise expression of cytokines is returning to normal levels during wound healing as for instance is shown for CCL2 and TNF-α[9]. Regarding ions it is reported that in rats during the first 5 days of normal healing after full thickness skin wounding Ca²⁺ concentrations are increasing to return to normal levels afterwards [10]. At day 5 after wounding also increased levels of Mg²⁺ and Zn²⁺ were found.

            2.1.2.  Burn Wounds:Persons with acute wounds including burn wounds are generally in a good healthy state at the moment of wounding. This is in contrast to patients with hard-to-heal wounds as discussed in the next chapter. Furthermore, in comparison to other acute wounds based on other impacts (e.g. cut, rupture) and also from hard-to-heal wounds, burn wounds behave different in several respects. Heat it selves induces a set of general effects. For instance, due to heat capillary permeability largely increased resulting in a significant plasma loss. This fluid flow is mostly stopped until 48h after thermal wounding. The wound fluid composition also differs from that of other wounds or blood serum promoting not only the healing process [11] due to the higher levels of angiogenin, but also inhibits (or at least diminish) proliferation of the wound contaminating bacteria [12]. Additionally, Nissen and co-workers found significant lower levels of fibroblast growth factor (FGF)-2 in burn relative to that of surgical wound fluid both being collected 6-12 h after injury [13]. Furthermore, especially if affected areas are large and deep (second-deep and third degree), immune system deregulations occur. These manifests as immunosuppression [14] in the beginning resulting in an increased likelihood of infection. Concomitant a hyper inflammatory response is seen with a chance of a shock [15,16]. This is not seen with other wounds. Forinstance, Mikhal’chik and co-workers [17]reported that before surgical treatment (around 4 days after burn) and relative to healthy children the blood plasma of children with uncomplicated burns exhibited increased concentrations of IL-6, IL-8 and MCP-1. In blood serum of children with complicated burns (septic toxaemia, toxaemia, and pneumonia) additionally the concentrations of IL-1RA, IL-10, TNF-α, IFN-γ and Granulocyte Colony-Stimulating Factor (GM-CSF) were increased.Plasma cytokine concentrations in the last group were generally much higher than in first group of patients. Interesting to note is that no correlation was found between the concentration of any of the evaluated 27 cytokines in blood plasma and exudate.

            2.1.3. Hard-to-Heal Wounds: In most cases wounds are able to heal rapidly and completely within weeks. However, in some cases they need, if at all, a very long time to heal, do very often never really close and/or wounds rapidly re-occur[18]. The latter are termed hard-to-heal or chronic wounds. Although so far, no official definition exists for hard-to-heal wounds [19], it is general accepted that these wounds fail to heal with standard therapy in an orderly and timely manner. This stagnation of the healing process may occur at any for the sequential phases of normal wound healing. Commonly, a prolonged strong reduction of blood and oxygen supply is the cause. The latter may be due on the one hand on bad vasculature. The latter may be evoked by disturbed transport of blood away from the involved area giving rise to venous ulcers or by insufficient transport to this area causing arterial (insufficiency) ulcers. On the other hand, reduced blood circulation may be based on external pressure (e.g. limited change of position in bed or wheel chair resulting in decubitus ulcers or pressure ulcers). People with diabetes mellitus have a prevalence to develop a pressure ulcer kind of ulcer that occur at the bottom of the feet and is termed Diabetic (Foot) Ulcer (DFU) [20]. The predisposition of diabetic in developing hard-to-heal wounds are based on various factors such as microcirculatory deficiencies [21], decreased Activated Protein C (APC) levels [22] or the presence of hyperglycaemia [23].

Several aspects may be mentioned that characterizes a chronic wound. One aspect is certainly the local tissue temperature which is increased at the wound site compared with peri-wound skin [24]. Increase of temperature is a sign of increased inflammation, critical microbial colonization or other factors disturbing the wound healing. As mentioned above, another characteristic of wounds is the production of wound fluid as reaction on tissue injury. The wound fluid is on the one hand defined by the amount that is produced per time unit and on the other hand by its pH and composition. Comparing acute normal healing and chronic wounds differences are seen in the change of wound fluid pH over time[25]. At wounding the acute wound pH is around 6.2 and rapidly go down to a pH below 6. During the inflammation phase the pH rises and reaches in the granulation phase a value above 7 during the granulation phase. During epithelization pH goes down again to around 6.2. In case of a hard-to-heal wound the latter decrease does not occur. It stays above pH 7.The pH has a large impact on wound healing mainly due to its effects on enzyme activity and bacterial colonization [25].

Besides the pH evolution also the wound fluid composition is different depending on the healing stage and wound type. It may be assumed that the wound fluid is directly reflecting the state of the wound bed physiology [26] and differs from that of the blood serum[27-29]. Most wound fluids of hard-to-heal wounds are characterized by increased proteolytic activity and increased levels of a large set of immune cells specific cytokines and other molecules (Table 1(A, B)).

Components that are released by bacteria (Table2). Non-healing may be associated with persistentstimulation of the innateimmuneresponse as suggested by Pukstad and co-workers for hard-to-heal venouslegulcers[37]. This may be based on the diseased state of the patient [39] and/or due to the presence of bacteria. Generally, a hard-to-heal wound is contaminated with bacteria. The inflammatory cells besides combatting the microbial contaminants also release proteases and actively degrade the provisional Extracellular Matrix (ECM) [40]. The latter is needed for starting tissue regeneration. Thus, inflammation seems to be a general aspect of the hard-to-heal wound. However, Liu and co-workers concluded after reviewing various publications regarding venous ulcer pathogenesis that many of the statements made do not withstand a critical examination [41]. For instance, against current opinion, they suggest that there is no proven clear indication that pro-inflammatory cytokines and growth factors are increased in hard-to-heal venous ulcers or that TGF-β and TNF-α are actively involved in the chronicity of the disease. Due to unclear patient inclusion-exclusion criteria of the studies, by not taking confounding factors into account (like age, gender, and wound aetiology) and/or too small number of patients the base on which the statements are funded is rather weak. The same may be true for other types of ulcers. As a result, there is certainly a need for new studies which take the criticised aspects into account.

2.2.  Bacterial Colonization and Its Effects on Wound Healing

Bacterial contamination of the wound is seen as a crucial factor in the delay of wound healing.It has been reported that more than 1000 bacterial species normally live on the human skin [51]. As a result, after wounding it is not a question if but a matter of time a wound gets contaminated by single planktonic bacteria. The bacterial composition of contaminated wounds is extremely complex and varies between patient and wound [52]. However, some similarities in bacterial composition seem to exist between the types of wounds. For instance, Acinetobacteria were mostly detected in acute wound infections of patients injured on the battle field [53]. This group of bacteria is also most prominent on normal skin [54]. In contrast, in hard-to-heal wounds (characterized by a chronic bacterial contamination) increased populations of Firmicutes (with Staphylococcus aureus as member) and Proteobacteria(e.g. Pseudomonas aeruginosa) and less Acinetobacteriaare observed [54]. A colonization of the wound by methicillin-resistant S. aureusand P. aeruginosaare suggested to delay wound healing [55]. The effect of products of P. aeruginosa(primarily Rhamnolipid B) is reported to inhibit bacterial clearance by polymorphonuclear leukocytes[43,56]. This may explain why low levels of P. aeruginosafacilitateS. aureusinfection in a rat model of complex orthopaedic wounds [57]. However, bacteria may not only be detrimental for wound healing. It has been reported that inoculation of a sterile wound with low levels of S. aureusor of P. aeruginosa may even promote wound healing[45,58].

A wound represents an optimal environment for bacterial colonization and proliferation: It is warm, moist and nutritious. These bacteria release, among others, a set of so called, quorum signalling molecules. Above a certain threshold level these molecules modify bacterial gene expression (quorum sensing, QS) and the excretion of biofilm matrix components starts. Embedded in this matrix bacteria are protected from attacks by the environment. After biofilm maturation planktonic bacteria are released that may start a new colony elsewhere or invade the tissue as premise for infection. Given the importance attributed to QS for gene regulation in bacteria [48]it may be interesting to note that significant differences in gene expression ofP. aeruginosain burn and hard-to-heal wounds are observed [59]. A variety of molecules are known to serve as QS molecules with different degree of specificity, i.e. from bacterial class to species specific. However, also other components which do not serve as signalling molecules are synthesized and released, produced as metabolites or which formation are catalysed by bacteria. Biogenic diamines and thiol compounds and other organic species may be related to bacterial degradation ofthe tissues [60]. Some examples are listed in Table 2. Depending on their vapour pressure a part may also vaporize. They are responsible for the malodour which is characteristic for increased anaerobic bacterial species colonizing the hard-to-heal wound [61]. Evidence was found that alterations in the overallVOC (Volatile organic compound) profile rather than quantitative analysis of single specific VOC species may give the greatest insight into hard-to-heal wound metabolic processes [60].

2.3.  Current Wound Management and Dressings

Burn and hard-to-heal wound management aims to stimulate wound closure by provide an optimal environment for healing, e.g. by giving optimal moisture balance, protect wound edges, remove dead cells, fibrin and biofilm by good debridement, and antimicrobial treatment[62-64]. Besides grafting of skin (temporary) skin substitutes in case of burn skin wounds [65], the currently used dressings and bandages focus predominantly on protection of the wound, exudate and moisture management, aid in debridement, combating bacterial contamination, reduction of malodour and wound pain[66-69]. Despite the proven positive effects of these a number of treatments [70] the closure of burn and hard-to-heal wounds still remain extremely challenging [65,71]. Since failure of treatment may result in amputation and morbidity, currently a next generation of dressings are under development or already on the market focussing additionally on promotion of vasculature for instance by vacuum therapy, improving wound oxygenation by additional hyperbaric oxygen pressure or by adding oxygen delivering dressings, on stimulating wound regeneration by steering the wound pH, by adding therapeutic agents including antibiotics and/ or on delivery of an (artificial) degradable extracellular matrix backbone as intermediate solution for improved tissue regeneration[4,25,67,69,72-76]. Although the latter dressings positively affect wound regeneration, they are not able to provide the wound manager with information what the actual state of the wound below the dressings is. However, large efforts are currently made in developing high-tech dressings including sensors with which at least some wound parameters can be monitored, such as moisture, pH, oxygen tension and/or temperature[3,77-79].

3.      Monitoring the State of Wounds by Means of Biosensors

In the last decade an increasing number of biosensors have been developed. Sensors can be classified according the technology used (optical, electrochemical, thermometric, piezoelectric or magnetic) or according the parameter which is measured.In relation to wounds these parameters are for instance temperature, moisture, pH, oxygen tension and wound fluid and volatile components including bacterial factors. In the next few chapters of each characteristic type some latest examples in biosensor development are given.

3.1.  Temperature

One characteristic of hard-to-heal wounds is the inflammation-based local increase in temperature.High temperature gradients between feet of diabetic type 2 patients may predict onset of neuropathic ulceration [80] or to predict healing of venous ulcers [24]. Some efforts were made to assess the surface temperature, on the one hand by infra-red images[24,81] The key limitations of the infra-red images are that the dressing has to be removed for this and the absence of on-line monitoring possibilities.Their advantage is certainly the high 2 D resolution. On the other hand, biosensors have been developed based on the use of thermistor materials (materials which resistance varies with temperature and the latter reproducible). These materials are usually films which can be made of metal (such as platinum [82] or gold [83] or carbon (graphite or multiwall Carbon Nanotubes (CNT)[84,85]. These biosensors make an on-line monitoring possible and organized in an array they deliver a low resolution 2 D picture of skin surface temperature[85]. To improve the flexibility of the sensor (array) recently an interesting concept was developed by Chen and co-workers[83]. The sensor is based on a highly meandering gold lane which is placed between water repellent breathable flexible semipermeable membranes with flexible CNT films for the wiring. The challenges of this kind of sensors still represent the signal to noise ratio, for obtaining a high-resolution picture of the temperature distribution the size of the sensors and the wiring organisation of the array of sensors.

3.2.  Moisture

For a good wound healing a correct moisture balance is needed. This is between insufficient, resulting in a drying out of the wound killing the ingrowing cells, and excessive, which may lead to tissue maceration.Various methodologies to measure moisture have been described[86,87], However, only one is so far implemented for moisture measurements in wound dressings. In 2013 a commercial product came on the market (WoundSense of Ohmedics) with which moisture condition at the wound site can be monitored. It is based on 2 parallel electrodes with a defined distance measuring the impedance between them[3,77](Figure 2A). Recently also another promising approach was described to assess moisture in bandages[88]. It makes use of the capability of graphene to bind water molecules. The latter affect its conductivity which can be measured[89].

3.3.  pH

The pH evolution of a wound is dependent on its state of healing and the phase of wound regeneration[25], the tissue type and bacterial colonization[93]. By that the pH cannot be used as sole parameter to assess the wound condition. Nevertheless, biosensors have been developed enabling a monitoring of the wound pH. For instance, a sprayable one which is based on the use of amino cellulose particles functionalized with fluorescein isothiocyanate as pH indicator and [ruthenium(II)-t ris-(4,7-diphenyl-1,10-phenanthroline as reference dye. pH pattern of the wound is visualized by processing pictures taken using two wavelengths, one to determine the pH-dependent luminescence of the pH indicator and one to relate this to the pH independent intensity of the reference dye[94].A limitation of this concept is its unknown long-term effect of the molecules used on the tissue (cytotoxicity and the inability for on-line monitoring. More applicable seem to be in this regards the concepts based on electrochemical measurements. For instance, recently Rahimi and co-workers described an inexpensive, flexible array of potentiometric kind(the difference in electrode potentials is measured) of pH sensors which can be integrated in a wound dressing [90](Figure 2B).

The operation of the pH sensors is based on the protonation and de-protonation of nitrogen atoms by the H⁺-ions of the wound fluid in the polymer chains of the polyaniline layer around the carbon electrode. The resulting change at the electrode surface charge relative to the reference electrode is taken as an index of wound fluid pH. One limitation of application is the period of use since KCl concentration of the reference electrode cover will assimilate to that of the wound fluid. It must be noted that a drift in signal was already seen after 5 h of use. Beside potentiometric also voltammetry (the cell's current is measured over time) methodologies have been pursued. A recent example is given by Mc Lister and Davis[95]. They used a 2-electrode based system composed of a silver chloride reference electrode and a poly-L-tryptophan modified carbon fibre-mesh electrode (Figure 3A).

3.4.  Oxygen Tension

The oxygen tension in wounds is dramatically reduced. For instance, in hard-to-heal wounds it is only in the range of 0.6-2.6% whereas that of normal subcutaneous in the range of 4-7%[103]. Different methods to measure oxygen tension have been described. Mostafalu and co-workers developed a bandage based on a flexible galvanic oxygen sensor to on-line monitor the wound oxygen tension [91](Figure 2C). The measurement is based on the reduction O₂ at a silver electrode delivering OH^- as product. At the same time a zinc atom of the zinc electrode is reduced forming Zn²⁺. The oxidation velocity is directly related to the oxygen concentration of the fluid that contacts the sensor. So far, the system has only been evaluated using a simulated wound set-up. The system has the potency to be organised as array but like all electrochemical set-up that were developed so far still large efforts must be put in the wiring concept and the miniaturization of the analysis device. Another type of sensor based on oxygen-dependent quenching of luminescence of metal porphyrin complexes has been described by Babilas and co-workers [104] and more recently by Wisniewski and co-workers [105]. With this immobilized in a polystyrene matrix as transparent planar sensor the surface pO₂ distribution can be mapped with a high temporal resolution of approximately 100 ms and a spatial resolution of at least 25 μm. The sensor as designed by Wisniewski and co-workers is integrated in an injectable tissue-integrating hydrogel with a size of 0.5X0.5X5 mm with which oxygen tension can be monitored for month to years inside the body.

3.5.  Wound Fluid Components Including Bacterial Factors

The concentration of large set of molecules is highly dependent on the state of the wound. These are not only released or modified by cells of the patients but might also be synthesized or produced as metabolites by bacteria. All of these represent potential diagnostic marker molecules characterizing the wound state. Furthermore, their change in concentration may be used as an indication for the impact of wound therapy. Regarding the detection of specific components in wound fluids two different philosophies are currently pursued. One is to analyse the fluid after sampling in a specialized laboratory or using a kind of a test strip, and this with smallest volume, lowest costs, increasing specificity and highest sensitivity. The other one is to analyse it continuously directly within wound dressings. It can be recognized that the methodologies which are developed for external analysis are used as base or toolbox for the development of biosensors which can be integrated in wound dressings. Therefore, both kinds of biosensors are mentioned below.

             3.5.1.   Dressing External Optical Analysis:Several optical biosensors have been proposed for the detection of biomarkers wound fluid samples. Here one can distinguish label-free and label-based set-ups. Label-Free Optical Set-Up. One example of a label-free set-up is the one proposed by Krismastuti and co-workers for the detection of bacterial colonization of hard-to-heal wounds[106]. It is based on the interferometric reflectance at the Nanoporous aluminium oxide surface due to difference in light path of laser in and outside the pores (Figure 5A). Here the pores are filled with a poly-lysine based substrate. The latter can be digested by proteinase K which is produced by P. aeruginosa. The digestion affects the light which entering the pores and this results in a shift of the interferometric reflectance pattern. The presence of a shift represents thus an indication for the bacterial presence. Another label-free set-up to detect bacteria is proposed by Sai and co-workers and is based on evanescent wave fibre optics (Figure 5B)[107]. Here antibodies against E. coliare bound to an optical fibre.Laser light is coupled into the fibre at one and light intensity is measured at the other end. E. coli binding changes the refractive index of the surface layer resulting in a coupling out of light and by that in a reduction of light intensity at the end of the fibre. The main disadvantage of the presented set-up is its low sensitivity. As third kind of method plasmon resonance-based set-up may be mentioned. For example, Sriram and co-workers used this technology to detect specific growth factors in fluids[108]. Here, a glass surface was coated with a thin layer of conductor (e.g. gold) and is brought in contact with a fluid. In the presence of laser light at the glass side plasmon waves are formed at the conductor- fluid interface region (Figure 5C).

Its oscillations interfere with the laser light, i.e., it can enhance and reduce its intensity depending on the angle at which it hits the surface.The plasmon waves are sensitive to the adsorption of molecules of the medium. As a result, the interference is changed and by that the surface plasmon resonance angle as well as the light intensity at a certain reflectance angle.A slightly more sensitive and advanced set-up (Figure 5D) was developed by Liu and co-workers [109] combining the principles of plasmon resonance (Figure 5C) and evanescent wave biosensor (Figure 5B). For this a flow chamber was built around a light-guiding silica capillary that is stripped off its cladding and coated with a 50-nm gold film. The latter gold film is functionalized with antibodies against the biomarker of interest. The whole is connected to a smartphone, taking images and monitoring the change of relative intensity as index of the biomarker concentration in the perfusate.

Label-Based Optical Set-Up. The simplest and most common label-based assays for all types of antigens are the ELISA test and immunofluorescence assay. Here biomarkers are quantified using specific antibodies which are linked to an enzyme catalysing the formation of a molecule with a specific absorbance, respectively, to a fluorochrome. The extent of absorption or fluorescence is directly related to the biomarker concentration of interest in the wound fluid. The immunofluorescence assay methodology was extended by using an evanescent set-up as described in figure 5B but labelling the antigen with a fluorochrome subsequently[110,111]. By being adsorbed the fluorochrome is located within the evanescence layer and excited as a result. Instead of measuring the light intensity at the end of the fibre, the fluorescence is measured which intensity is again reflecting the antigen concentration.

A more complex set-up is developed by Thet and Jenkins [100] for detecting bacterial toxins. Their sensor is based on toxin-sensitive vesicles which are filled with a fluorochrome (Figure 6A).Due to the presence of the toxin the vesicles break and the fluorochrome is released. As a result, the fluorescence intensity of the solution will increase.

Enzymes as biomarkers offer the possibility to take advantage of their characteristic to alter structures and to take the latter as index for their concentration. Here, specific moieties are connected to fluorochrome units supressing its fluorescence capabilities. These moieties are designed in a way that they can be cleaved (removed) by the specific biomarker enzyme of interest. As a result, it becomes fluorescent again (Figure 6B). For example, Hasmann and co-workers developed optical sensors using this principle for assessing the activity of the serine protease neutrophil elastase (HNE) and cathepsin G (CatG) in the wound fluid[112]. These proteases are involved in the pathogenesis of a number of inflammatory disorders and are seen as markers for early infection.Another way of detection offers the linking of two different chromophores which in the linked situation deliver a FRET (Förster resonance energy transfer) signal (Figure 6C).

An enzymatic cleavage of this link increases the distance between the two resulting in the disappearance of the FRET phenomenon. This kind of set-up was used by Schulenberg and co-workers to quantify HNE activity[113]. Similarly, redox-responsive (and even reversible) near infrared small-molecule biosensors have been proposed which is based on the change in fluorescence intensity due to a redox induced structural change of the fluorochrome [114](Figure 6D). On the one hand, since these chromophores as described are not bound to a surface and/or the assay is a multistep approach, these set-ups as proposed cannot be included as such as monitoring system in dressings. But even if so, the proposed set-up’s (except the previous one) are designed for single measurement use and a fluorescence analysing unit would have to be included in the dressing to be able to monitor fluorescence or FRET intensity speaking against the use of such sensor inside dressings. On the other hand, there are also clear advantages of these above mentioned-label-based optical set-ups for biomarker detection [113,115]. These assays are reported to be not only specific and sensitive but also can be done relatively quickly.

               3.5.2.  Dressing External Electrode-Based Analysis

Different kind of electrode-based biosensors have been developed (Figure 2D, 3, 4). For instance, a quick test for small wound fluid samples has been proposed for the detection of some key markers for inflammation (TREM-1, MMP-9 and bacterial HSL) [116]. This is done using Faradaic 3 electrode electrochemical impedance spectroscopy (for an in-depth description of the methodology see [98](Figure 3D). For this antibodies against the marker molecule are bound to the working electrode and the change in impedance properties is reported to be related to the quantity of bound marker molecules. Because electrodes have to be treated with wound fluid before impedance measurement (for which a non-biocompatible Fe(CN)₆]^3−/4−redox system is used) this set-up cannot as such be implemented for the use inside a wound dressing.A similar system is used by Henihan and co-workers for detecting bacterial rRNA[99](Figure 3E). Here, Peptide Nucleic Acid (PNA) probe for the targeted rRNA is bound to the working electrode and after treating with solution containing the target rRNA again the change in impedance properties is measured.In this case the impedance characteristics are modified by the degree of hybridisation. A variant of this method was described by Thet and Jenkins for detecting bacterial contamination (Figure 3F). They developed a 3-electrode electrochemical sensor concept for the detection of virulence factors from S. aureusand P. aeruginosa. It is also based on the use of non-biocompatible K₃[Fe(CN)₆] but here biomimetic vesicles are containing it[100]. In the presence of toxins like rhamnolipids and delta toxin the vesicles are damaged. The released content can be quantified by a change in redox current. An interesting new miniature biosensor approach for detecting ultralow quantities of biomarker molecules (1 pg/ml) in lowest volume samples was reported by Tlili and co-workers[92]. It is based on connecting two gold electrodes with a single walled carbon nanotube which is functionalised with an antibody against the biomarker of interest (in this study cortisol) (Figure 2D). In the presence of the biomarker the resistance is found to be concentration dependently strongly reduced. So far, this sensor has not been adapted for and evaluated with wound fluids. For analysing fluid compositions also, field effect transistors have been developed which are similar as the one shown in figure 4 (and to a certain degree the example shown in figure 2D) but with an analyte-specific modification of the dielectric gate insulator such as a specific antibody, enzyme or other reactant[102,118,117]. Unfortunately, all of these sensors are for single use only and not made for integration in wound dressings. It may be noted that the sensor platforms such as presented in figures 2D and 3A, D, E and 4 are strongly dependent on the design of the electrode surfaces (in case of field effect transistor: of the dielectric gate isolator outer surface) and its specificity to react with solely the biomarker of interest. In the meantime, numerous surfaces have been made and their sensitivity tested (See:[117,119,120]), However, although a high sensitivity (taking detection limit as index) could be shown for most applications, challenges which still remain is the interference with other components of the fluid including biofouling effects and reliable scalable fabrication methods for mass production of these sensors [117].

                3.5.3.Dressing Internal Optical Analysis

The optical biosensors designed to be included in dressings are to a large extend similar to the ones used for external analysis. The difference is that the fluorochrome is more or less linked to the wound dressing. For instance, like the set-up used by Thet and Jenkins [100](Figure 6A), Zhou and co-workers [121] used toxin-sensitive vesicles filled with fluorochrome to detect bacterial contamination. In case of Zhou and co-workers these vesicles were attached to a fabric as representative for a dressing. Also, here the fluorochrome is released by the vesicles as result of the presence of toxins/virulence factors. The resulting increased fluorescence is visualized using a high intensity UV source and after taking an image the latter is analysed. Its applicability and specificity were proven using S. aureusand P. aeruginosa cultures with E.colias non-pathogenic reference but unfortunately not its biocompatibility. The latter is of course of key importance since the fluorochrome is released into the wound. The latter disadvantage is not present if the fluorochrome is directly coupled to the wound dressing. For instance, Derikvand and co-workers [122] (like the set-up used by Hasmann and co-workers [112](Figure 6B) used moiety tethered fluorochromes and bound them to dressing components such as cellulose. Only in case these moieties were enzymatically removed (in this example by esterases) the fluorochrome gets fluorescent which is visualized like the previous one using an UV source.

Although by coupling the fluorochrome to the dressing one disadvantage (possible cytotoxic effects) could be eliminated, others are still not solved. Like the dressing external optical biosensors, the disadvantage of these kind of sensors is that it is only applicable if the marker enzyme is negligible present in the correctly healing wound but which concentration is strongly increased in case the dressing has to be changed or if wound needs additional therapy. Furthermore, fluorescence has to be judged by eye (or from an image taken from the wound or dressing) and is not quantified by an equipment that is integrated in the dressing.

                3.5.4. Dressing Internal Electrode-Based Analysis

Several electrode-based sensors have been proposed to be integrated in wound dressings or which integration is possible. One example is the one developed by Sharp and Davis for sensing bacterial wound contamination taking urate as an index[96]. Urate is normally present in the wound at concentrations of 190-420µM and bacteria, especially P. aeruginosa, are able to metabolise it resulting in a significant wound fluid concentration reduction[96]. Their biosensor with a two-electrode configuration is to a certain degree similar to the one described for measuring pH (Figure 2B). However, in this case the conductive proton selective polymer is omitted and instead of measuring at one specific voltage voltammogram are made in the range of -0.4 to +1.2 V (Figure 3B). The wound fluid urate is quantified by the typical shape of the square wave voltammogram taking the maximum electric flow in µAmpere around 0.2 V as index. Similarly, but using 3 electrodes with a bias potential of 350mV and measuring the resulting current, Liu and Lillehoj could reproducibly monitor urate concentrations [123]. Unfortunately, both set-up was only tested using different urate concentrations in simulated body fluids but were not evaluated under real conditions using wound fluids.

Another example of electrode-based sensor is described by Farrow and co-workers for measuring bacterial load of the wound fluid[97]. With their sensor (Figure 3C) impedance profiles are assessed during frequency sweeps from 1 MHz to 0.1 Hz at a root mean square voltage of 200 mV. With this they were able to correlate the obtained pattern with the bacterial load. Similar has been described by Sheybani and Shukla [124]. However, unfortunately both were only evaluated using artificial fluids and again not using wound fluids. Other examples of optical and electrode-based biosensors and their limitations for the specific detection of pathogenic microorganisms and other parameters can be found among others in reviews of Silva and co-workers[125]Damborsky and co-workers [126] and by You and Lee[127].

3.6.  Odour

So far malodour’s are analysed and characterized using large external devices such chromatography-mass spectrometry[60]. To circumvent this disadvantage Lu and co-workers developed a promising electrochemical set-up in which electrodes were functionalized with odorant-binding proteins from the honeybee[128]. Binding of the odorant resulted in a different impedance spectrum.Recently, Dieffenderfer and co-workers developed another and new kind of small mass-based biosensor for volatile compounds in the exhaled air of asthma patients. It is founded on the use of a capacitive micro-machined ultrasonic transducer which acts as an electrostatically actuated mechanical resonator. Thanks to its nature, specific volatile compound(s) adhere to the coating resulting in a change of mass bound and by that in a change in mechanical resonant frequency. Its principle can probably also be used for wound volatile compounds. Thanks to its reduced size it may be integrated into wound dressings in future.

4.      Knowledge Gaps

Wounds are complex and the kind and state of the wound determines which treatment is optimal. For a proper classification multiple parameter have to be evaluated. Although significant progress has been made in the development of biosensors for wound monitoring, the development of high-tech sensors for wound dressings is still in its infancy stage. This may be traced back on various knowledge gaps of which some are mentioned below:

(i)                            A solid knowledge base is still missing regarding molecules which may be used as sensor key target representatives. Not all reported studies can withstand a critical evaluation and should be interpreted with care. Furthermore, the representatives should not only enable an early prognosis regarding the fate of the wound with the applied therapy but also recognize early changes as effect of additional or changed therapy.

(ii)                          One key factor of most of the current biosensors is that the analysis of obtained images or wound fluids/exudate cannot be performed at the point-of-care but still must be done in a laboratory with often long assay times. Here new wearable microdetectors and/or rapid analysing units have to be developed that uses a different kind of detection method enabling a (nearly) on-line monitoring.

(iii)                         The state of the wound differs within the wound. Therefore, it is important to get a high resolution 2D picture of the state of the wound. With some exceptions, current sensors are too large for this and if miniaturized the wiring represent a challenging issue.

(iv)                        Another key limiting factor for the use of most above-mentioned biomarker molecule biosensors is the fact that they can only be used once (and by that can deliver only a single snapshot of the current state) and are not able to monitor the wound state. The latter is especially relevant for targeting declining marker component concentrations or which concentrations elevation is relative small relative to normal levels. For this, biosensors are needed that are able to regenerate between the measurements. Some possible strategies are listed in a review of Goode and co-workers[129].

(v)                          Limited specificity and sensitivity as well as biofouling are for some biosensors key limiting factors and ways have to be found to solve this [117]. Furthermore, many of the proposed set-up were only evaluated using artificial fluids and not under real conditions, i.e. on the wound or using wound fluids.

(vi)                        Material inertness a key issue, especially for biosensors based on materials that are sprayed into the wound or which are known to release constituents. Unfortunately, for many compounds the long-term biological effects are not known.

(vii)                       The delayed healing of wounds is considered as a disease symptom. Therefore, knowledge of the disease, the underlying mechanism by which they induce hard-to-heal wounds and the relation between disease therapy and wound healing process are of key importance. However, in most cases this is only rudimentary known and in addition partially patient (and by that wound) specific. With the development of new biosensors this knowledge will increase which on their turn will help to improve biosensor target definitions.

The above-mentioned knowledge gaps represent currently to our opinion the key bottle necks in the development of sensitive and prognostic biosensors for high-tech wound dressings. Therefore, filling these white pages should have first priority as premise for the development of the next generation high-tech wound dressings.

5.      Foresight of Future High-Tech Dressings

The removal of the above-mentioned bottlenecks certainly represents a are big challenge for the future. However, the reward will be commensurate. The list below represents certainly only a tip of potential improvements which could be foreseeable.

5.1.  Telemedicine

Since tethered monitoring technology is cumbersome to the patient and also because it strongly restricts the behaviour of the patients, wireless solutions may be preferred. This also enables remote patient monitoring of the patients living at home (Figure 7A). In case wound dressings needs to be replaced or treatment has to be adapted an alert will be given to the patient and practical nurse. This will enable a therapy or wound management intervention at the correct time point ensuring that care givers do not interact with patients more than they otherwise would, but also that a critical change in wound state is not missed. Additionally, based on the wound values the correct diagnosis can be given and evolved treatment initiated on the earliest possible stage. As consequence life quality of the patient may be increased as well as costs and resources may be saved. Currently, telemonitoring is already discussed for wound care and other health issues[79,130-133].

5.2.  Holistic Approaches

Since the way wound heals is directly affecting the extent of scar formation, dressings will be developed that addresses wound closure and scar formation. For instance, to address the effect of increased tension on scar formation promotion, future dressings may include force modulating properties to control the wound mechanical environment[134,135].

5.3.  Personalized Medicine -Patient Specific Dressings

The need for personalised medicine and based on this patient specific dressings is unquestioned and will be common in future. First steps in this direction are made. For instance, it could be shown that an adaptation of the wound care based on the characterization of the bioburden by molecular diagnostics could significantly improve the treatment outcome [136]. Not only the bacterial background also differences in wound healing based on the genetic background of the patient and based on the prevalence for the development of certain disorders may require a personalised approach for wound treatment. For instance, for therapeutics that are metabolized by the CYP2D6 or CYP2C19 gene product a test came on the market as an aid to clinicians to analyse of the CYP2D6 and CYP2C19 genotype of the patient in order to adapt accordingly therapeutic strategy and treatment dose for these therapeutics[137].

5.4.  Theranostics

Wound therapeutic wound dressings will become available which include on the one hand biosensors sensing the wound state and on the other hand therapeutic compounds. The capability of dressings to release specific therapeutic compounds depending on certain most common pathological wound progress patterns, would have a tremendous potential for most optimal point-of-care wound care management (regarding time point, location of treatment and compound dose) (Figure 7B). These can be like the sensor proposed by Zhou and co-workers for bacterial detection [121] but instead of releasing a fluorochrome a drug or antibiotic would be released. The premise for this is beside the availability of a complete set of highly specific markers, the availability of a dense high-resolution biomonitoring and data analysis unit. Furthermore, the development of a set of controllable matrices of miniaturized compound release units for the local delivery of the various compounds is crucial enabling to redirect wound healing process from the not preferred /unwanted pattern towards the rapid healing state.

6.      Acknowledgements

The critical reading of the manuscript by Dr.Qun Ren and Dr. Guido Panzarasa (Empa, Switzerland) is greatly acknowledged

 A.      Venous ulcers

Table 1: Some wound fluid components which are significantly reduced or increased to a relevant extent in non-healing diabetic foot ulcer and venous leg ulcer wound.

1.       Smith, Nephew (2016) The true costs of wounds. In: Management, W. (Ed.), Hull, UK.

2.       Dowsett C, Bielby A, Searle R (2014) Reconciling increasing wound care demands with available resources. J Wound Care 23: 552-558.

3.       Milne SD, Seoudi I, Al Hamad H, Talal TK, Anoop AA, et al. (2016) A wearable wound moisture sensor as an indicator for wound dressing change: an observational study of wound moisture and status. Int Wound J 13: 1309-1314.

4.       Contardi M, Heredia-Guerrero JA, Perotto G, Valentini P, Pompa PP, et al. (2017) Transparent ciprofloxacin-povidone antibiotic films and nanofiber mats as potential skin and wound care dressings. Eur J Pharm Sci 104: 133-144.

5.       DaCosta RS, Kulbatski I, Lindvere-Teene L, Starr D, Blackmore K, et al. (2015) Point-of-care autofluorescence imaging for real-time sampling and treatment guidance of bioburden in chronic wounds: first-in-human results. PLoS One 10: e0116623.

6.       Salvo P, Dini V, Di Francesco F, Romanelli M (2015) The role of biomedical sensors in wound healing. Wound Medicine 8: 15-18.

7.       Texier I, Marcoux P, Pham P, Muller M, Benhamou PY, et al. (2014) SWAN-iCare: A smart wearable and autonomous negative pressure device for wound monitoring and therapy. Wireless Mobile Communication and Healthcare (Mobihealth). IEEE, Agios Konstantinos, Greece. Pg No: 137-144.

8.       GrimstadØ, SandangerØ, Ryan L, Otterdal K, Damaas JK, et al. (2011) Cellular sources and inducers of cytokines present in acute wound fluid. Wound Repair Regen 19: 337-347.

9.       Mori R, Power KT, Wang CM, Martin P, Becker DL (2006) Acute downregulation of connexin43 at wound sites leads to a reduced inflammatory response, enhanced keratinocyte proliferation and wound fibroblast migration. J Cell Sci 119: 5193-2003.

10.    Lansdown ABG, Sampson B, Rowe A (1999) Sequential changes in trace metal, metallothionein and calmodulin concentrations in healing skin wounds. J Anat 195: 375-386.

11.    Pan SC, Wu LW, Chen CL, Shieh SJ, Chiu HY (2012) Angiogenin expression in burn blister fluid: implications for its role in burn wound neovascularization. Wound Repair Regen 20: 731-739.

12.    Gonzalez MR, Fleuchot B, Lauciello L, Jafari P, Applegate LA, et al. (2016) Effect of human burn wound exudate on Pseudomonas aeruginosa virulence. mSphere 1: e00111-00115.

13.    Nissen NN, Gamelli RL, Polverini PJ, DiPietro LA (2003) Differential angiogenic and proliferative activity of surgical and burn wound fluids. J Trauma 54: 1205-1210.

14.    Horgan AF, Mendez MV. O'Riordain DS, Holzheimer RG, Mannick JA, et al. (1994) Altered gene transcription after burn injury results in depressed T-lymphocyte activation. Ann Surg 220: 342-351.

15.    Daniel T, Thobe BM, Chaudry IH, Choudhry MA, Hubbard WJ, et al. (2007) Regulation of the postburn wound inflammatory response by gammadelta T-cells. Shock 28: 278-283.

16.    Holzheimer RG, Molloy R, Mendez MV, O'Riordain D, Curley P, et al. (1995) Multiple system organ failure may be influenced by macrophage hypoactivation as well as hyperactivation-importance of the double challenge. Eur J Surg 161: 795-903.

17.    Mikhal'chik EV, Piterskaya JA, Budkevich LY, Pen'kov LY, Facchiano A, et al. (2009) Comparative study of cytokine content in the plasma and wound exudate from children with severe burns. Bull ExpBiol Med 148: 771-775.

18.    Lazarus GS, Cooper DM, Knighton DR, Margolis DJ, Pecoraro RE, et al. (1994) Definitions and guidelines for assessment of wounds and evaluation of healing. Arch Dermatol 130: 489-493.

19.    Streit M, Mayer D, Traber J (2012) Definition von Wunden: Akute und chronischeWunden. Wund Management 3: 4-10.

20.    Graves N, Zheng H (2014) The prevalence and incidence of chronic wounds: a literature review. Wound Practice Res 22: 4-19.

21.    Chao CYL, Cheing GLY (2009) Microvascular dysfunction in diabetic foot disease and ulceration. Diabetes/Metab. Res Rev 25: 604-614.

22.    Whitmont K, Fulcher G, Reid I, Xue M, McKelvey K, et al. (2013) Low circulating protein C levels are associated with lower leg ulcers in patients with diabetes. BioMed Res Int 2013: 719570.

23.    Berlanga-Acosta J, Schultz GS, López-Mola E, Guillen-Nieto G, García-Siverio M, et al. (2013) Glucose toxic effects on granulation tissue productive cells: the diabetics’ impaired healing. BioMed Res Int 2013: 256043.

24.    Nakagami G, Sanada H, Iizaka S, Kadono T, Higashino T, et al. (2010) Predicting delayed pressure ulcer healing using thermography: a prospective cohort study. J Wound Care 19: 465-472.

25.    Schneider LA, Korber A, Grabbe S, Dissemond J (2007) Influence of pH on wound-healing: a new perspective for wound-therapy? Arch Dermatol Res 298: 413-420.

26.    Widgerow AD, King K, Tussardi IT, Banyard DA, Chiang R, et al. (2015) The Burn Wound Exudate - an under-utilized resource. Burns 41: 11-17.

27.    Ambrosch A, Lobmann R, Pott A, Preissler J (2008) Interleukin-6 concentrations in wound fluids rather than serological markers are useful in assessing bacterial triggers of ulcer inflammation. Int Wound J. 5: 99-106.

28.    Gohel MS, Windhaber RA, Tarlton JF, Whyman MR, Poskitt, KR (2008) The relationship between cytokine concentrations and wound healing in chronic venous ulceration. J VascSurg 48: 1272-1277.

29.    Trengove NJ, Langton SR, Stacey MC (1996) Biochemical analysis of wound fluid from nonhealing and healing chronic leg ulcers. Wound Repair Regen 4: 234-239.

30.    Löffler M, Schmohl M, Schneiderhan-Marra N, Beckert S (2011) Wound fluid diagnostics in diabetic foot ulcers In: Dinh, T. (Ed.), Wound Fluid Diagnostics in Diabetic Foot Ulcers, Global Perspective on Diabetic Foot Ulcerations. Intech, Rijeka, Croatia.

31.    Weigelt C, Rose B, Poschen U, Ziegler D, Friese G, et al. (2009) Immune mediators in patients with acute diabetic foot syndrome. Diabetes Care 32: 1491-1296.

32.    Liu Y, Min D, Bolton T, NubéV, Twigg SM, et al. (2009) Increased matrix metalloproteinase-9 predicts poor wound healing in diabetic foot ulcers. Diabetes Care 32: 117-119.

33.    Krisp C (2013) Proteomic profiling of exudates from diabetic foot ulcers and acute wounds using mass spectrometry. p. 183. Macquarie Uni, Sydney.

34.    Fernandez ML, Upton Z, Edwards H, Finlayson K, Shooter GK (2012) Elevated uric acid correlates with wound severity. Int Wound J 9: 139-149.

35.    Hoffmann DC, Textoris C, Oehme F, Klaassen T, Goppelt A, et al. (2011) Pivotal role for alpha1-antichymotrypsin in skin repair. J BiolChem 286: 28889-28901.

36.    Beidler SK, Douillet CD, Berndt DF, Keagy BA, Rich PB, et al. (2008) Multiplexed analysis of matrix metalloproteinases in leg ulcer tissue of patients with chronic venous insufficiency before and after compression therapy. Wound Repair Regen 16: 642-648.

37.    Pukstad BS, Ryan L, Flo TH, Stenvik J, Moseley R, et al. (2010) Non-healing is associated with persistent stimulation of the innate immune response in chronic venous leg ulcers. J Dermatol Sci 59: 115-122.

38.    Eming SA, Koch M, Krieger A, Brachvogel B, Kreft S, et al. (2010) Differential proteomic analysis distinguishes tissue repair biomarker signatures in wound exudates obtained from normal healing and chronic wounds. J Proteome Res 9: 4758-4766.

39.    Xu F, Zhang C, Graves DT (2013) Abnormal cell responses and role of TNF-α in impaired diabetic wound healing. BioMed Res Int 2013: 754802.

40.    Wilgus TA, Roy S, McDaniel JC (2013) Neutrophils and wound repair: Positive actions and negative reactions. Adv. Wound Care (New Rochelle) 2: 379-388.

41.    Liu YC, Margolis DJ, Isseroff RR (2011) Does inflammation have a role in the pathogenesis of venous ulcers? A critical review of the evidence. J Invest Dermatol 131: 818-827.

42.    Labows JN, McGinley KJ, Webster GF, Leyden JJ 1(980) Headspace analysis of volatile metabolites of Pseudomonas aeruginosa and related species by gas chromatography-mass spectrometry. J ClinMicrobiol 12: 521-526.

43.    Jensen PØ,Bjarnsholt T, Phipps R, Rasmussen TB, Calum H, wet al. (2007) Rapid necrotic killing of polymorphonuclear leukocytes is caused by quorum-sensing-controlled production of rhamnolipid by Pseudomonas aeruginosa. Microbiol 153: 1329-1338.

44.    Sharp D, Gladstone P, Smith RB, Forsythe S, Davis J (2010) Approaching intelligent infection diagnostics: Carbon fibre sensor for electrochemical pyocyanin detection. Bio electrochemistry 77: 114-119.

45.    Kanno E, Kawakami K, Miyairi S, Tanno H, Suzuki A, et al. (2016) Promotion of acute-phase skin wound healing by Pseudomonas aeruginosa C4 -HSL. Int. Wound J 13: 1325-1335.

46.    Nakagami G, Sanada H, Sugama J, Morohoshi T, Ikeda T, et al. (2008) Detection of Pseudomonas aeruginosa quorum sensing signals in an infected ischemic wound: an experimental study in rats. Wound Repair Regen 16: 30-36.

47.    Sifri CD (2008) Healthcare epidemiology: quorum sensing: bacteria talk sense. Clin Infect Dis 47: 1070-1076.

48.    Goryachev AB (2009) Design principles of the bacterial quorum sensing gene networks. Wiley Interdiscip. Rev SystBiol Med 1: 45-60.

49.    Xavier KB, Bassler BL (2003) LuxS quorum sensing: more than just a numbers game. CurrOpinMicrobiol 6: 191-197.

50.    Kong KF, Vuong C, Otto M (2006) Staphylococcus quorum sensing in biofilm formation and infection. Int J Microbiol 296: 133-139.

51.    Pappas S (2009) Your Body Is a Wonderland ... of Bacteria. Science. NOW Daily News.

52.    Bowler PG, Duerden BI, Armstrong DG (2001) Wound microbiology and associated approaches to wound management. ClinMicrobiol Rev 14: 244-269.

53.    Be NA, Allen JE, Brown TS, Gardner SN, McLoughlin KS, et al. (2014) Microbial profiling of combat wound infection through detection microarray and next-generation sequencing. J ClinMicrobiol 52: 2583-2594.

54.    Han A, Zenilman JM, Melendez JH, Shirtliff ME, Agostinho A, et al. (2011) The importance of a multifaceted approach to characterizing the microbial flora of chronic wounds. Wound Repair Regen 19: 532-541.

55.    Bowling FL, Jude EB, Boulton AJ (2009) MRSA and diabetic foot wounds: contaminating or infecting organisms? Curr Diab Rep 9: 440-444.

56.    Bjarnsholt T, Kirketerp-Møller K, Jensen PØ, Madsen KG, et al. (2008) Why chronic wounds will not heal: a novel hypothesis. Wound Repair Regen 16: 2-10.

57.    Hendricks KJ, Burd TA, Anglen JO, Simpson AW, Christensen GD, et al. (2001) Synergy between Staphylococcus aureus and Pseudomonas aeruginosa in a rat model of complex orthopaedic wounds. J Bone Joint Surg Am 83-A: 855-861.

58.    Carrel A (1921) Cicatrization of wounds: XII. Factors initiating regeneration. J Exp Med 34: 425-434.

59.    Turner KH, Everett J, Trivedi U, Rumbaugh KP, Whiteley M (2014) Requirements for Pseudomonas aeruginosa acute burn and chronic surgical wound infection. PLoS Genet 10: e1004518.

60.    Thomas AN, Riazanskaia S, Cheung W, Xu Y, Goodacre R, et al. (2010) Novel noninvasive identification of biomarkers by analytical profiling of chronic wounds using volatile organic compounds. Wound Repair Regen 18: 391-400.

61.    Bowler PG, Davies BJ, (1999) The microbiology of infected and noninfected leg ulcers. Int J Dermatol 38: 573-578.

62.    Leaper DJ, Schultz G, Carville K, Fletcher J, Swanson T, et al. (2012) Extending the TIME concept: what have we learned in the past 10 years? Int. Wound J 9 Suppl 2: 1-19.

63.    Stojadinovic A, Carlson JW, Schultz GS, Davis TA, Elster EA (2008) Topical advances in wound care. GynecolOncol 111: S70-S80.

64.    Raposio E, Bortolini S, Maistrello L, Grasso DA (2017) Larval Therapy for Chronic Cutaneous Ulcers: Historical Review and Future Perspectives. Wounds 29: 367-373

65.    Sterling JP, Heimbach DM, Gibran NS (2010) Management of the burn wound. In: Hamilton, O.N. (Ed.), Trauma and thermal injury. Decker Intellectual Properties, Denver.

66.    Fogh K, Andersen MB, Bischoff-Mikkelsen M, Bause R, Zutt M, (2012) Clinically relevant pain relief with an ibuprofen-releasing foam dressing: results from a randomized, controlled, double-blind clinical trial in exuding, painful venous leg ulcers. Wound Repair Regen 20: 815-821.

67.    Fonder MA, Lazarus GS, Cowan DA, Aronson-Cook B, Kohli AR, et al. (2008) Treating the chronic wound: A practical approach to the care of nonhealing wounds and wound care dressings. J Am AcadDermatol 58: 185-206.

68.    Lipman RD, van Bavel D (2007) Odor absorbing hydrocolloid dressings for direct wound contact. Wounds 19: 138-146.

69.    Martin C, Low WL, Amin MC, Radecka I, Raj P, et al. (2013) Current trends in the development of wound dressings, biomaterials and devices. Pharm Pat Anal 2: 341-359.

70.    Frykberg RG, Banks J (2015) Challenges in the treatment of chronic wounds. Adv. Wound Care 4: 560-582.

71.    Nielsen J, Fogh K (2015) Clinical utility of foam dressings in wound management: a review. Chronic Wound Care Management Res 2: 31-38.

72.    Boateng J, Catanzano O, (2015) Advanced therapeutic dressings for effective wound healing - A review. J Pharm Sci 104: 3653-3680.

73.    Saab IR, Sarhane KA, Ezzeddine HM, Abu-Sittah GS, Ibrahim AE (2014) Treatment of a paediatric patient with a distal lower extremity traumatic wound using a dermal regeneration template and NPWT. J Wound Care 23: S5-58.

74.    Baldursson BT, Kjartansson H, Konrádsdóttir F, Gudnason P, Sigurjonsson GF, et al. (2015) Healing rate and autoimmune safety of full-thickness wounds treated with fish skin acellular dermal matrix versus porcine small-intestine submucosa: a noninferiority study. Int J Low Extrem Wounds 14: 37-43.

75.    Seow WY, Salgado G, Lane EB, Hauser CA (2016) Transparent crosslinked ultrashort peptide hydrogel dressing with high shape-fidelity accelerates healing of full-thickness excision wounds. Sci Rep 6: 32670.

76.    Shao M, Hussain Z, Thu HE, Khan S, de Matas M, et al. (2017) Emerging Trends in Therapeutic Algorithm of Chronic Wound Healers: Recent Advances in Drug Delivery Systems, Concepts-to-Clinical Application and Future Prospects. Crit Rev Ther Drug Carrier Syst 34: 387-452.

77.    McColl D, MacDougall M, Watret L, Connolly P (2009) Monitoring moisture without disturbing the wound dressing. Wounds (UK) 5: 94-99.

78.    Puchberger-Enengl D, Krutzler C, Binder M, Rohrer C, Schröder KR, et al. (2012) Characterization of a multi-parameter sensor for continuous wound assessment. Procedia Engineering 47: 985-988.

79.    Wood C (2015) DermaTrax smart dressing makes wound care less intrusive and more efficient. New Atlas.

80.    Armstrong DG, Holtz-Neiderer K, Wendel C, Mohler MJ, Kimbriel HR, et al. (2007) Skin temperature monitoring reduces the risk for diabetic foot ulceration in high-risk patients. Am J Med 120: 1042-1046.

81.    Dini V, Salvo P, Janowski FD, Barbini A, Romanelli M (2015) Correlation between wound temperature obtained with an infrared camera and clinical wound bed score in venous leg ulcers. Wounds 27: 274-278.

82.    Moser Y, Gijs MAM (2007) Miniaturized flexible temperature sensor. JMicroeltromech. Syst 16: 1349-1354.

83.    Chen Y, Lu B, Chen Y, Feng, X (2015) Breathable and stretchable temperature sensors inspired by skin. Sci Rep 5: 11505.

84.    Matzeu G, Losacco M, Parducci E, Pucci A, Dini V, et al. (2011) Skin temperature monitoring by a wireless sensor. IECON 2011 - 37th Annual Conference on IEEE Industrial Electronics Society. IEEE, Melbourne, VIC, Australia. Pg No: 3533 - 3535.

85.    Shih WP, Tsao LC, Lee CW, Cheng MY, Chang C, et al. (2010) Flexible temperature sensor array based on a graphite-polydimethylsiloxane composite. Sensors 10: 3597-3610.

86.    Cancilla PA, Barrette P, Rosenblum F (2002) On-line moisture determination of ore concentrates 'a review of traditional methods and introduction of a novel solution'. Mol Cell Probes 16: 393-408.

87.    Yeo TL, Sun T, Grattan KTV (2008) Fibre-optic sensor technologies for humidity and moisture measurement. Sensors Actuators 144: 280-295.

88.    MailOnline (2016) Girl, 13, wins $15,000 after inventing moisture-sensing bandage that tells doctors when it needs to be changed. Health, October 4, 2016 ed. Associated Press, USA.

89.    Wee BH, Khoh WH, Sarker AK, Lee CH, Hong JD (2015) A high-performance moisture sensor based on ultralarge graphene oxide. Nanoscale 7: 17805-17811.

90.    Rahimi R, Ochoa M, Parupudi T, Zhao X, Yazdi IK, et al. (2016) A low-cost flexible pH sensor array for wound assessment. Sensors Actuators B 229: 609-617.

91.    Mostafalu P, Lenk W, Dokmeci MR, Ziaie B, Khademhosseini A, et al. (2015) Wireless flexible smart bandage for continuous monitoring of wound oxygenation. IEEE Trans. Biomed Circuits Syst 9: 670-677.

92.    Tlili C, Myung NV, Shetty V, Mulchandani A (2011) Label-free, chemiresistor immunosensor for stress biomarker cortisol in saliva. BiosensBioelectron 26: 4382-4386.

93.    Gethin G (2007) The significance of surface pH in chronic wounds. Wounds UK 3: 52-56.

94.    Schreml S, Meier RJ, Weiß KT, Cattani J, Flittner D, et al. (2012) A sprayable luminescent pH sensor and its use for wound imaging in vivo. ExpDermatol 21: 951-953.

95.    McLister A, Davis J (2015) Molecular wiring in smart dressings: Opening a new route to monitoring wound pH. Healthcare (Basel) 3: 466-477.

96.    Sharp D, Davis J (2008) Integrated urate sensors for detecting wound infection. Electrochem. Comm 10: 709-713.

97.    Farrow MJ, Hunter IS, Connolly P (2012) Developing a real time sensing system to monitor bacteria in wound dressings. Biosensors 2: 171-188.

98.    Katz E, Willner I (2003) Probing biomolecular interactions at conductive and semiconductive surfaces by impedance spectroscopy: Routes to impedimetric immunosensors, DNA-sensors, and enzyme biosensors. Electroanalysis 15: 913-947.

99.    Henihan G, Schulze H, Corrigan DK, Giraud G, Terry JG, et al. (2016) Label- and amplification-free electrochemical detection of bacterial ribosomal RNA. BiosensBioelectron 81: 487-494.

100.Thet NT, Jenkins ATA (2015) An electrochemical sensor concept for the detection of virulence factors from Staphylococcus aureus and Pseudomonas aeruginosa. Electrochem. Comm 59: 104-108.

101.Duroux P, Emde C, Bauerfeind P, Francis C, Grisel A, Thybaud L, et al. (1991) The ion sensitive field effect transistor (ISFET) pH electrode: a new sensor for long term ambulatory pH monitoring. Gut 32: 240-245.

102.Lee CS, Kim SK, Kim M (2009) Ion-sensitive field-effect transistor for biological sensing. Sensors 9: 7111-7131.

103.Schreml S, Szeimies RM, Prantl L, Karrer S, Landthaler M, et al. (2010) Oxygen in acute and chronic wound healing. Br J Dermatol 163: 257-268.

104.Babilas P, Liebsch G, Schacht V, Klimant I, Wolfbeis OS, et al. (2005) In vivo phosphorescence imaging of pO₂ using planar oxygen sensors. Microcirculation 12: 477-487.

105.Wisniewski NA, Nichols SP, Gamsey SJ, Pullins S, Au-Yeung KY, et al. (2017) Tissue-integrating oxygen sensors: Continuous tracking of tissue hypoxia. Adv Exp Med Biol 977: 377-383

106.Krismastuti FS, Bayat H, Voelcker NH, Schönherr H (2015) Real time monitoring of layer-by-layer polyelectrolyte deposition and bacterial enzyme detection in nanoporous anodized aluminum oxide. Anal Chem 87: 3856-3863.

107.Bharadwaj R, Sai VV, Thakare K, Dhawangale A, Kundu T, Titus S, et al. (2011) Evanescent wave absorbance based fiber optic biosensor for label-free detection of E. coliat 280 nm wavelength. BiosensBioelectron 26: 3367-3370.

108.Sriram R, Yadav AR, Mace CR, Miller BL (2011) Validation of arrayed imaging reflectometry biosensor response for protein-antibody interactions: cross-correlation of theory, experiment, and complementary techniques. Anal Chem 83: 3750-3757.

109.Liu Y, Liu Q, Chen S, Cheng F, Wang H, et al. (2015) Surface plasmon resonance biosensor based on smart phone platforms. Sci Rep 5: 12864.

110.Hsieh MC, Chiu YH, Lin SF, Chang JY, Chang CO, et al. (2015) Amplification of the signal intensity of fluorescence-based fiber-optic biosensors using a Fabry-Perot resonator structure. Sensors 15: 3565-3574.

111.Saaski EW (2009) BioHawk portable pathogen detector. In: Fauchet, P.M. (Ed.), Frontiers in Pathogen Detection: From Nanosensors to Systems, San Francisco.

112.Hasmann A, Gewessler U, Hulla E, Schneider KP, Binder B (2011) Sensor materials for the detection of human neutrophil elastase and cathepsin G activity in wound fluid. ExpDermatol 20: 508-513.

113.Schulenburg C, Faccio G, Jankowska D, Maniura-Weber K, Richter M (2016) A FRET-based biosensor for the detection of neutrophil elastase. Analyst 141: 1645-1649.

114.Chu TS, Lü R, Liu BT (2016) Reversibly monitoring oxidation and reduction events in living biological systems: recent development of redox-responsive reversible NIR biosensors and their applications in in vitro/in vivo fluorescence imaging. BiosensBioelectron 86: 643-655.

115.Janegitz BC, Cancino J, Zucolotto V (2014) Disposable biosensors for clinical diagnosis. J NanosciNanotechnol 14: 378-389.

116.Ciani I, Schulze H, Corrigan DK, Henihan G, Giraud G, et al. (2012) Development of immunosensors for direct detection of three wound infection biomarkers at point of care using electrochemical impedance spectroscopy. BiosensBioelectron 31: 413-418.

117.Ahmad R, Mahmoudi T, Ahn MS, Hahn YB (2018) Recent advances in nanowires-based field-effect transistors for biological sensor applications. BiosensBioelectron 100: 312-325.

118.Kaisti M (2017) Detection principles of biological and chemical FET sensors. BiosensBioelectron 98: 437-448.

119.Maduraiveeran G, Sasidharan M, Ganesan V (2018) Electrochemical sensor and biosensor platforms based on advanced nanomaterials for biological and biomedical applications. BiosensBioelectron 103: 113-129.

120.Adzhri R, Arshad MK, Gopinath SC, Ruslinda AR, Fathil MF, et al. (2016) High-performance integrated field-effect transistor-based sensors. Anal Chim Acta 917: 1-18.

121.Zhou J, Tun TN, Hong SH, Mercer-Chalmers JD, Laabei M, et al. (2011) Development of a prototype wound dressing technology which can detect and report colonization by pathogenic bacteria. BiosensBioelectron 30: 67-72.

122.Derikvand F, Yin DT, Brumer H (2016) Cellulose-based biosensors for esterase detection. Anal Chem 88: 2989-2993.

123.Liu X, Lillehoj PB (2017) Embroidered electrochemical sensors on gauze for rapid quantification of wound biomarkers. BiosensBioelectron 98: 189-194.

124.Sheybani R, Shukla A (2016) Complementary sensors for rapid and sensitive detection of wound bacteria. Conf. Proc. IEEE Eng Med BiolSoc 2016: 1902-1905.

125.Silva RR, Avelino KY, Ribeiro KL, Franco OL, Oliveira MD, et al. (2014) Optical and dielectric sensors based on antimicrobial peptides for microorganism diagnosis. Front Microbiol 5: 443.

126.Damborský P, Švitel J, Katrlik J (2016) Optical biosensors. Essays Biochem 60: 91-100.

127.Yoo SM, Lee SY (2016) Optical biosensors for the detection of pathogenic microorganisms. Trends Biotechnol 34: 7-25.

128.Lu Y, Li H, Zhuang S, Zhuang D, Zhang Q, et al. (2014) Olfactory biosensor using odorant-binding proteins from honeybee:Ligands of floral odors and pheromones detection by electrochemical impedance. Sensors Actuators B 193: 420-427.

129.Goode JA, Rushworth JVH, Millner PA (2015) Biosensor regeneration: A review of common techniques and outcomes. Langmuir 31: 6267-6276.

130.Clifford GD, Clifton D (2012) Wireless technology in disease management and medicine. Annu Rev Med 63: 479-492.

131.Mehmood N, Hariz A, Templeton S, Voelcker NH (2015) A flexible and low power telemetric sensing and monitoring system for chronic wound diagnostics. Biomed. Eng. Online 14: 17.

132.Ohmedics (2016) Advanced diagnostics from Bioelectronics for home, clinic and telehealth use.

133.Ye J, Zuo Y, Xie T, Wu M, Ni P, et al. (2016) A telemedicine wound care model using 4G with smart phones or smart glasses: A pilot study. Medicine (Baltimore) 95: e4198.

134.Corr DT, Hart DA (2013) Biomechanics of Scar Tissue and Uninjured Skin. Adv. Wound Care (New Rochelle) 2: 37-43.

135.Gurtner GC, Dauskardt RH, Wong VW, Bhatt KA, Wu K, et al. (2011) Improving cutaneous scar formation by controlling the mechanical environment: large animal and phase I studies. Ann Surg 254: 217-225.

136.Dowd SE, Wolcott RD, Kennedy J, Jones C, Cox SB (2011) Molecular diagnostics and personalised medicine in wound care: assessment of outcomes. J Wound Care 232: 234-239.

137.Roche Molecular Diagnostics, 2016.