Abstract:
Electrochemical biosensing devices and methods detect the activity of proteases and active infection in biological samples. The devices and methods utilize distance constraints between a redox reporter and an electrode to create a change in current that is detected. The distance constraints are released when an analyte containing a specified protease or proteases reacts with a protein substrate sequence in the analyte. The particular protease or proteases to be detected can be selected by using biosensors with particular substrate sequences. The devices and methods are not only qualitative, they can be used to quantitatively evaluated protease content in samples.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application claims the benefit of U.S. Provisional Application No. 62/065,440, filed Oct. 17, 2014. 
     
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
       [0002]    Not Applicable 
       THE NAMES OF PARTIES TO A JOINT RESEARCH AGREEMENT 
       [0003]    Not Applicable 
       INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC 
       [0004]    Not Applicable 
       BACKGROUND OF THE INVENTION 
       [0005]    1. Field of the Invention 
         [0006]    The invention broadly relates to electrochemical biosensing, more specifically to an electrochemical biosensing device to detect the activity of proteases and active infection in biological fluids and tissues, and even more particularly to a method of electrochemical biosensing to detect the activity of proteases and active infection in biological fluids and tissues. 
         [0007]    2. Description of the Related Art 
         [0008]    Differentiated from acute wounds that heal in a matter of days or weeks, chronic wounds persist for months or years and, may, last a lifetime. Such chronic wounds are commonly referred to as non-healing wounds. Examples include diabetic foot ulcers, venous leg ulcers, and pressure ulcers, and other types of chronic wounds including arterial ulcers, non-healing surgical wounds, burns, fistulae, dermatitis or vasculitis wounds, skin cancers, and radiation wounds. Unlike acute wounds that progress through the four normal phases of healing (hemostasis, inflammation, proliferation, and remodeling), chronic wounds stall in their healing, often in the inflammatory phase. Wounds fail to heal because molecular and cellular abnormalities in the wound prevent healing progress. 
         [0009]    Over the past decade, expert panels have met at international wound meetings to report the importance of effective assessment and diagnosis in the treatment of wounds. The first such international group of experts met in June, 2007, with the intent that their report would serve as a platform to stimulate the development of dedicated test kits that would beneficially influence the future management of problem wounds. This panel concluded that specific biochemical tests were urgently needed that would identify the causes of delayed healing in wounds that are slow-to-heal and that the most appropriate markers to serve as biochemical indicators of stalled wound healing would include elevated proteases, infection, and perhaps decreased nitric oxide. 
         [0010]    A second panel of experts of international recognition convened in February, 2011, and confirmed the need for biochemical wound diagnostics and identified elevated wound proteases as an important biochemical target. This panel also determined that an easy to use, point-of-care protease diagnostic would be of immense value in clinical practice. A third expert panel also convened in May, 2011, and concluded that wound protease levels would be a useful marker to help direct clinicians to specific treatments. The proteases most often elevated in non-healing wounds that are often stalled in an inflammatory state include matrix metalloproteinases, especially collagenases and gelatinases, and serine proteases, especially neutrophil serine proteases. The literature indicates that multiple matrix metalloproteinases (MMPs) are associated with stalled wound healing. These proteases are normal contributors to the early inflammatory phase of wound healing, to degrade damaged tissue and help prepare a clean wound bed for healing. But this activity normally occurs within three to five days after which the activities of these proteases reduce to very low levels in the wound environment. However, in non-healing wounds elevated levels of proteases persist and they continue to degrade extracellular matrix, growth factors and receptors involved in wound healing. This causes the wound not to fully heal. 
         [0011]    Multiple matrix metalloproteinases contribute to stalled wound healing including the gelatinases, MMP-2, and MMP-9; the collagenases MMP-1, MMP-8, and MMP-13; stromelysin, MMP-3; and matrilysin, MMP-7. Whereas some researchers tried to identify one of these to provide the best target for diagnostic test development, other researchers have proposed that the measurement of just one individual protease would be unlikely to be representative of the proteolytic environment of the wound. Although there have now been 25 MMPs described and classified, partly on historical assessment of the substrate specificity of the MMP (and partly on the cellular localization of each), it has become increasingly clear that there is a lot of overlap in their substrate specificities. This is not surprising since it has been shown that the structures of the catalytic domains of many MMPs revealed that their polypeptide folds are essentially superimposable. Furthermore, it has been shown that substrate specificities that might exist in their natural substrates further break down when short peptide substrates are used, due to the loss of secondary and tertiary structure that only exists in the full-length natural protein substrates. Peptide substrates have been identified that can be cleaved by multiple wound MMPs, and these promiscuous substrates would make appropriate reagents for a more representative determination of the total proteolytic activity in the wound environment. 
         [0012]    In addition to the presence of elevated MMP proteases in a wound, a large body of work has also identified that infection of wounds is also a major factor that prevents them from healing. Common wound pathogens include, but are not limited to,  Staphylococcus aureus  (including MRSA) and  Pseudomonas aeruginosa.  These bacteria exist in two major forms: either free-living single cells known as planktonic form, or as biofilm communities. Whereas planktonic bacteria are relatively easy to control (unless they consist of superbugs such as MRSA [Methicillin-resistant  Staphylococcus aureus ], C. Diff [ Clostridium difficile ], VRE [Vancomycin-resistant  Enterococci ], CRE [Carbapenem-resistant Enterobacteriaceae] and CRKP [Carbapenem-resistant  Klebsiella  pneumonia]), bacterial biofilms are much more difficult to remove. 
         [0013]    A biofilm is an assemblage of microbial cells that is irreversibly associated with a surface and is most often enclosed in a matrix of primary polysaccharide material often referred to as ‘slough’ in chronic wounds. Biofilm-associated organisms differ from their planktonic counterparts in the genes that are transcribed, resulting in an altered resistance to antibiotics and the human immune system. A biofilm colony is more similar to a multicellular organism with cell-cell interaction and communication and division of labor, and this makes such colonies very difficult to kill and control. In a biofilm colony there are some bacteria that are growing and reproducing and making the matrix they live in, while other cells exist in a protective, sprawling state that are relatively quiescent, but in the event of a catastrophic challenge, those cells can survive and re-seed the biofilm. For example, biofilm can often reestablish within twenty four (24) hours following removal by surgical debridement. 
         [0014]    Biofilm in wounds has become a significant problem. It has been estimated that up to sixty percent (≧60%) of non-healing wounds contain biofilm but it is difficult to diagnose. Some researchers suggest measuring wounds for the presence of common wound bacteria, but others point out that just as they are present on normal skin surfaces all over the human body, commensal bacteria also exist on wound surfaces that are not causing active infection. A diagnostic that cannot distinguish between an active infection and a passive microbial colonization is of little use as an indicator of the state of the wound with respect to whether the presence of microbes is preventing the wound from healing. 
         [0015]    As used herein, an ‘active’ infection is an infection caused by microorganisms, whether planktonic or associated in biofilm communities, that are maintaining a chronic state of wound inflammation or contributing to wound deterioration and preventing wound healing. Other suggestions for detecting the presence of biofilm include measuring components of the biofilm matrix, including polysaccharide, proteinaceous, or nucleic acid components. Such measurements, however, miss active infection caused by planktonic bacteria, and there are other problems with using such targets. 
         [0016]    There is a need for an invention that recognizes that a better indicator of active infection than bacteria or their constituents is a measurement of the stressful response of the host to the presence of active infection. In response to a microbial infection, the host mounts a rapid multi-faceted immune response. For the purposes of the present invention, this includes the sending of neutrophil granulocytes, which are an abundant type of white blood cells adept at killing and phagocytosing microorganisms and other foreign material. Neutrophils (also known as leukocytes) are one of the first responders of inflammatory cells to migrate towards sites of inflammation. They migrate through the blood vessels, then through interstitial tissue, following chemical signals from pro-inflammatory cytokines such as Interleukin-1 (IL-1), IL-8, and tumor necrosis factor-alpha (TNF-α), in a process called chemotaxis. While some have suggested the detection of the pro-inflammatory cytokines as suitable targets for the diagnosis of infection, there is still a need to measure the activity of host proteases as a more informative analyte. This is because the host proteases released from neutrophils are closest to describing the effects of infection because they are the specific entities performing the tissue damage. In addition, the concentrations of cytokines are much lower and therefore more difficult to detect and interpret. 
         [0017]    When neutrophils encounter a bacterial cell or foreign body, they become activated where they first initiate an oxidative attack against the bacterial cell wall and then release granules from their cytoplasm containing high concentrations of proteases into the bacteria through the hole in its outer cell membrane caused by the oxidative burst. These neutrophil proteases lead to the death of the pathogen by breaking down critical proteins inside the bacteria. Neutrophils also recognize biofilm as foreign and mount their attack against it, but the bacteria can ‘hide’ within the biofilm matrix, inaccessible to and protected from both the oxidative burst and the neutrophil proteases. The chronic presence of the biofilm colony causes the host&#39;s immune system to continually send neutrophils to try to remove it, resulting in high local concentrations of proteases in the wound, released from the activated neutrophils, and these cause significant damage to host tissue. 
         [0018]    This process occurs in non-healing (chronic) wounds causing degradation of newly developing extracellular matrix, but it also occurs in other host tissues resulting in multiple disease states including, but not limited to, chronic obstructive pulmonary disease, COPD, including chronic bronchitis and emphysema, and periodontitis (destruction of gum tissue). The most notable proteases contributing to this tissue damage by the host&#39;s own attack response are the neutrophil serine proteases (NSPs) secreted from neutrophil azurophilic granules including neutrophil elastase, proteinase-3, and cathepsin G. 
         [0019]    Accordingly, there is a long-felt need for a device and method for detecting the activity of proteases and active infection in biological fluids and tissues. 
       BRIEF SUMMARY OF THE INVENTION 
       [0020]    An object of the present invention is to provide an electrochemical biosensing device for detecting both elevated proteases and active infections in biological fluids and tissues by controlling distance constraints between a redox reporter and a biosensor electrode. 
         [0021]    Another object of the present invention is to provide a method of electrochemical biosensing detection of both elevated proteases and active infections in biological fluids and tissues by controlling distance constraints between a redox reporter and a biosensor electrode. 
         [0022]    A general object of the present invention is to detect and provide a quantitative measurement of both elevated proteases and active infections in chronic, non-healing wounds. 
         [0023]    It is yet another object of the present invention to provide an electrochemical biosensor to detect both elevated proteases and active infections in chronic, non-healing wounds. 
         [0024]    The use of the present invention is most importantly demonstrated in patients with non-healing wounds containing protease activities higher than normal. Such patients typically have weekly appointments with their wound care providers, and the point-of-care diagnostic test of the present invention will be administered at each patient visit to indicate whether the protease activity level has increased, stayed the same, or decreased since the last visit. By quantitatively measuring the protease activity level, the present invention indicates the efficacy of the chosen treatment strategy over time. If the elevated protease activity has decreased since the previous visit, the wound care provider and the patient are encouraged that the adopted treatment strategy is successfully contributing to the conversion of the non-healing wound onto a healing trajectory. If the protease activity level has not decreased since the last patient visit, or if it has increased, it would indicate to the wound care provider that the treatment strategy needs to be adjusted. Through this quantitative analysis, the present invention provides early, accurate point-of-care information to help guide the week-by-week management of the wound. By providing a quantitative measurement of wound protease activity at each patient visit, the present invention offers a unique advantage for the navigational direction of wound management. 
         [0025]    There are currently several therapeutic dressings designed to lower wound proteases. These include but are not limited to collagen/oxidized regenerated cellulose (e.g. Promogran by Systagenix), EDTA (e.g. Biostep by Smith &amp; Nephew), and PHI (e.g. Tegaderm Matrix by 3M) dressings. The present invention indicates whether one of these or other protease-lowing therapies is beneficial to use, and furthermore, after some time of use, whether the chosen therapy is effectively lowering the wound protease activity levels. The present invention indicates in a more-timely manner than the presently used subjective observation by the wound care provider, whether the treatment strategy is working or should be changed to another protease-lowering therapy. Also, the present invention indicates when additional treatment strategies, described below, not indicated when wound protease levels are elevated, can be incorporated in the management strategy. 
         [0026]    For example, present-day advanced wound therapies include the use of growth factors (e.g. Becaplermin [Regranex] platelet-derived growth factor by Smith &amp; Nephew) and skin substitutes (e.g. Apligraf and Dermagraft, both by Organogenesis) that are both expensive. Both growth factor and skin substrate therapeutic modalities are readily susceptible to proteolysis, such that if they are applied to wounds still containing high protease activities, they will be destroyed before providing any therapeutic effect. The present invention provides an important tool to wound care providers contemplating wound therapy using either growth factors or skin substitutes (living skin equivalent or LSE). The present invention provides to wound care providers, for the first time, a reliable quantitative measurement of wound protease activity to indicate whether the wound is receptive to growth factor or LSE treatment. This quantitative information provides an important corollary to designing and justifying subsequent wound treatments as required by the majority of insurance corporations. 
         [0027]    A further object of the invention is to detect active infection. Active infection is detected by the host stress response as determined by the release of large amounts of serine proteases from neutrophils (NSPs) recruited to inflamed and diseased tissue sites via chemotaxis. Specifically sites of infection recruit blood neutrophils, which release neutrophil serine proteases (NSPs) to attack the pathogenic organisms contributing to the infection. In an example embodiment, the measurement of these NSPs using the methods of the present invention indicate the host&#39;s stress level to the presence of active infection, and provide quantitative information to guide the infection management strategy. Infection therapeutic strategies including antibiotic administration and antibiofilm management are available, and are indicated by the elevated levels of NSPs by the present invention. 
         [0028]    Electrochemical detection using biosensors in various configurations has been proposed for the detection of specific target molecules involved in multiple biological processes, including the diagnosis of disease. Most widely known is the electrochemical detection and monitoring of blood glucose for the management of diabetes. Electrochemical detection has also been proposed for the detection of other analytes including antibodies in whole blood; cocaine and other drug detection in biological fluids; proteases such as thrombin, trypsin and plasmin, endopeptidase neurotoxins, prostate specific antigen, MMP-3 as a cancer biomarker and MMP-7; cytokines, including interferon (IFN)-γ and tumor necrosis factor (TNF)-α; and detection of unpurified PCR amplification products of microbial pathogen DNA. 
         [0029]    Detecting methods include, but are not limited to, potentiostatic measurement via either voltammetric methods, such as alternating current voltametry, linear sweep voltametry, cyclic voltametry, square wave voltametry, differential pulse voltametry, normal pulse voltametry, differential normal pulse voltametry, or amperometric methods, such as amperometric detection, fast amperometry, pulsed amperometric detection, and zero resistance amperometry. Alternatively, a galvanostatic measurement system could be used, such as linear sweep potentiometry, cyclic potentiometry, potentiometric detection (galvanostatic), fast potentiometry, zero current potentiometry, potentiometric stripping analysis, either galvanostatic or faradaic. 
         [0030]    The invention can be applied for the detection and quantitation of elevated protease activity and active infection within wounds, as indications of stalled healing in chronic wounds such as diabetic foot ulcers, venous leg ulcers, pressure ulcers, arterial ulcers, wounds of mixed etiology, burns, non-healing surgical wounds and others. But the present invention is also applicable to other disease states involving elevated proteases resulting from a host stress response, such as gingivitis and periodontitis, chronic obstructive pulmonary disease (e.g. emphysema or chronic bronchitis), and other disorders. 
         [0031]    These and other objects and advantages of the present invention will be readily appreciable from the following description of preferred embodiments of the invention and from the accompanying drawings and claims. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0032]    The nature and mode of operation of the present invention will now be more fully described in the following detailed description of the invention taken with the accompanying drawing figures, in which: 
           [0033]      FIG. 1  is a computer generated model of lifting peptide SEQ ID NO: 48, an α-helix  102  with the attachment cysteine on the bottom of the figure at the amino-terminus  101  and promiscuous MMP substrate SEQ ID NO: 3 constituting a open loop  103  at the carboxyl-terminal end of the construct at the top of the figure. This model was generated using PEP-FOLD where SEQ ID NO: 48 was predicted to be an a-helix in most models, in agreement with its native structure in apolipoprotein E. 
           [0034]      FIG. 2  is a first embodiment of an electrochemical biosensor according to the invention utilizing a peptide-oligonucleotide conjugate bound to a signaling oligonucleotide, showing before and after protease cleavage. 
           [0035]      FIG. 3  is a second embodiment of an electrochemical biosensor according to the invention utilizing an aptamer recognition peptide bound to a signaling aptamer, showing before and after protease cleavage. 
           [0036]      FIG. 4  is a third embodiment of an electrochemical biosensor according to the invention utilizing an oligonucleotide-peptide-oligonucleotide conjugate bound to a signaling oligonucleotide, showing before and after protease cleavage. 
           [0037]      FIG. 5  is a front view of the electrical meter of the diagnostic device of the present invention. 
           [0038]      FIG. 6  is a front view of a diagnostic test strip of the diagnostic device that could be used with the electrical meter depicted in  FIG. 5 ; the test strip uses manual addition of a specific volume of biological sample. 
           [0039]      FIG. 7  is an exploded view of a diagnostic test strip of the diagnostic device used with the electrical meter depicted in  FIG. 5 ; the test strip uses automatic addition of a specific volume of biological sample. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0040]    At the outset, it should be appreciated that like drawing numbers on different drawing views identify identical, or functionally similar, structural elements of the invention. While the present invention is described with respect to what is presently considered to be the preferred aspects, it is to be understood that the invention as claimed is not limited to the disclosed aspects. 
         [0041]    Furthermore, it is understood that this invention is not limited to the particular methodology, materials and modifications described and as such may, of course, vary. It is also understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to limit the scope of the present invention, which is limited only by the appended claims. 
         [0042]    Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs. It should be appreciated that the term “sample” is synonymous with terms such as “fluid”, “detection device” is synonymous with terms such as “meter”, and such respective terms may be used interchangeably as appearing in the specification and claims. Although any methods, devices or materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods, devices, and materials are now described. 
         [0043]    Adverting now to the figures,  FIG. 1  is a computer-generated structure of a lifting peptide based on SEQ ID NO:  48 ,  102 , which holds MMP and/or NSP peptide substrate sequence  103  above a biosensor electrode in an accessible position for proteolysis, and ultimately lifts a redox reporter above the biosensor electrode preventing current from flowing prior to proteolysis by sample proteases. This ‘signal-on’ configuration is represented in  FIG. 2 . 
         [0044]    Two other lifting mechanisms are shown in  FIGS. 3 and 4 , where the protease cleavage sites  303  and  403 , respectively, and redox reporters  304  and  404 , respectively, are initially held above the working electrode  601  or  709  in a ‘signal-on’ configuration by either an aptamer recognition lifting peptide  302  bound to a signaling aptamer  305 , or by a double-stranded DNA constituted by an oligonucleotide-peptide-oligonucleotide conjugate  402  bound to a signaling oligonucleotide  405 , respectively. 
         [0045]    As shown in  FIGS. 1-7 , the present invention provides for a quantitative measurement of protease activity using a small and reproducible quantity of a biological sample  702 .  FIGS. 5-7  illustrate the electrical meter  502  and diagnostic test strips  600  and  700  that use an electrochemical biosensor, either  200 ,  300 , or  400 , which is free of optical detection limitations, and is based on a distance constraint mechanism to hold a redox reporter  204 ,  304 , or  404  above an electrode  206 ,  306 , or  406  prior to proteolysis by sample proteases. The molecular architecture provides for three example embodiments: 1) a lifting peptide-oligonucleotide conjugate (PepOliCon)  202  bound to a signaling oligonucleotide  205 , shown in  FIG. 2 ; 2) a lifting peptide coupled to a specific aptamer-recognition peptide sequence (aptamer epitope)  302  bound to a signaling aptamer  305 , shown in  FIG. 3 ; and 3) a lifting double-stranded DNA structure constituted by a portion of an oligonucleotide-peptide-oligonucleotide (OligoPepOligo) conjugate  402  and a complementary portion of a signaling oligonucleotide  405 , shown in  FIG. 4 . All three embodiments provide the mechanism to lift the respective redox reporter  204 ,  304 , or  404  on the terminus of the signaling oligonucleotide/aptamer  205 ,  305 , or  405  high enough above the electrode  206 ,  306 , or  406  of the electrochemical biosensor  200 ,  300 , or  400  so as to initially inhibit current flow through the working electrode connection  605  or  708 . Although  FIG. 5  illustrates units of measurement as micrograms per milliliter (μg/mL), other measurement units are acceptable. The electrical meter  502  measures the protease activity as a concentration when diagnostic test strip  600  or  700  is inserted into slot  501  and has a biological sample  702  applied to the diagnostic test strip  700 . 
         [0046]      FIG. 6  shows an example configuration of a test strip  600  that is commercially available from multiple suppliers and which could be used in the present invention. This test strip configuration (constituted by a working electrode  601 , a counter-electrode  602 , and a reference electrode  603 , with respective connectors  605 ,  606 , and  604  that make contact inside electrical meter  502  when correctly inserted into slot  501 ) would require manual sample acquisition and application of an accurate amount of sample to working electrode  601 . Such accurate sample acquisition and application could be made by a micropipette or capillary tube, or such other method as would be known to one skilled in the art. 
         [0047]      FIG. 7  shows the structure of test strip  700 . Test strip  700  is similar to that described by Alva 2008, includes multiple layers and could be custom-made for the present invention. The following description of test strip  700  in  FIG. 7  starts at the top layer  704  and progresses downward to the bottom layer that contains electrochemical biosensor  709  and working electrode connector  708 . The top layer  704  of test strip  700  includes the initializing bar  705 . The next layer includes sample volume indicator electrodes  706  and reference electrode  703 . Underneath this is a spacer layer  707  with a calibrated sample acquisition channel  701  of controlled dimensions. Sample channel slot  701  within spacer  707  draws a reproducible volume of biological sample  702  via capillary action over the biosensor  709  to facilitate the reaction between proteases in the sample  702  and the reagents on the biosensor  709 . Underneath spacer  707  is the bottom layer, containing the electrochemical biosensor  709  working electrode where the chemistry takes place. Proteolytic cleavage of the biosensor reagent allows the redox tag to collide with the electrode surface (as shown in  FIGS. 2-4 ), producing a readily detectable Faradaic current (ΔE) that travels down a connector of the working electrode  708  to the electrical meter  500 . The electrochemical biosensor utilizes an electrode strip  206 ,  306 , or  406 , which can be constructed of various materials, including but not limited to, gold, platinum, carbon, nation-coated carbon, and polymers of such materials. The chemical reaction takes place on the electrode strip  206 ,  306 , or  406  when there are proteases in the biological sample. The resulting faradaic current (ΔE) is directly proportional to the concentration of active proteases in the sample and therefore provides an accurate quantification of sample protease activity. 
         [0048]    As depicted in  FIG. 7 , the orientation of the multiple layers of test strip  700  are strategically aligned for the detection process. For instance, initializing bar  705  is located at one end of test strip  700  for insertion into detection input slot  501  of the electrical meter  502  depicted in  FIG. 5 . When test strip  700  is placed into detection input slot  501  of the electrical meter  502 , initializing bar  705  turns on the electrical meter  502 . The opposing end of test strip  700  includes sample channel slot  701 , into which biological sample  702  is drawn by capillary action to be deposited above electrochemical biosensor  709 . 
         [0049]    A volume of biological sample  702  is controlled by dimensions of the sample channel slot  701 . The electrochemical biosensor  601  and  709 , as chemically constituted, are shown in  FIGS. 2-4 . Filling of the sample channel slot  701  is detected electronically using the sample volume indicator electrodes  706  and starts the reaction time, after which the flowing faradaic current is read on the detection device  502  as an indication of protease activity in the biological sample  702 . If biological sample volume is inadequate, the reaction time is not initiated and electrical meter  502  displays an error message, such as “inadequate sample volume”. To eliminate any measurement errors, a new test strip  700  is typically used after an error message. 
         [0050]    The chemical reaction to detect protease levels in the biological sample  702  occurs on the electrochemical biosensor  709 . Respective embodiments of the electrochemical bionsensor include the PepOliCon electrochemical biosensor  200 , the aptamer electrochemical biosensor  300 , or the OligoPepOligo electrochemical biosensor  400 . The electrochemical biosensor  709  is prepared for use by attaching the reagents described herein. For the example embodiment illustrated in  FIG. 2 , the peptide-oligonucleotide conjugate (PepOliCon)  202  and the signaling oligonucleotide  205  are mixed together resulting in a binding interaction between their respective complementary DNA sequences. Then, the duplex (PepOliCon  202  bound to signaling oligonucleotide  205 ) is attached via covalent bonding to the biosensor electrode  206  via their terminal sulfhydryl groups  201 . 
         [0051]    As shown in  FIG. 2 , the lifting peptide keeps the redox reporter  204 , an example of which is methylene blue, positioned far enough above the biosensor electrode  206  so as not to produce a faradaic current flowing through the biosensor electrode  206  and subsequently the working electrode connection  605  or  708 . Once the reagents  202  and  205  are covalently attached on the biosensor electrode  206 , the electrochemical biosensor  200  is ready to receive a biological sample. Biological sample is either pipetted manually onto the working electrode  601  if a test strip such as that depicted in  FIG. 6  is used, or is gathered automatically over the biosensor working electrode  709  via capillary action into the sample channel slot  701  of test strip  700 , which overlays the electrochemical biosensor  709  that includes the reagent duplex PepOliCon  202  bound to signaling oligonucleotide  205 . The lifting peptide moiety of the PepOliCon  202  holds the redox reporter  204  above the biosensor electrode  206  in the ready position prior to cleavage of the substrate cleavage sequence by proteases in the biological sample, thereby preventing electrical current from flowing through the working electrode connection  605  or  708  while the biosensor is in the ‘ready’ configuration. 
         [0052]    If proteases are present in the biological sample, the proteases cleave a specific substrate sequence  203  that is a part of the PepOliCon construct; examples of such protease substrate sequences are disclosed herein as SEQ ID NOs: 1-47. Once cleaved, the DNA portion  207  of the PepOliCon construct is released from the lifting effect of the lifting peptide moiety  202  of the PepOliCon such that the redox reporter  204  collides with the surface of the biosensor electrode  206 , thereby causing the flow of electrical current. The lifting peptide moiety of the PepOliCon  202 , by its molecular structure, acts like a tensioned spring holding the redox reporter  204  above the biosensor electrode  206  surface. Once cleaved, the effect of the lifting tension is lost thereby allowing the redox reporter  204  to bend towards the biosensor electrode  206  surface; the more protease that is present in the biological sample, the more PepOliCon is cleaved on the electrochemical biosensor  200 . Therefore, the amount of current flow is directly proportional to how much protease is present in the biological sample. 
         [0053]    The lifting mechanism of biosensor  300  is also mediated by a peptide  302 , whereas that of biosensor  400  is mediated via a length of double-stranded DNA consisting of portions of reagents  402  and  405 . 
         [0054]    In each embodiment depicted in  FIGS. 2-4 , a sample protease-induced hydrolysis at the top of either a lifting peptide or a lifting double-stranded DNA breaks the attachment between either the lifting peptide of biosensors  200  and  300  or the lifting double-stranded DNA of biosensor  400  and the signaling oligonucleotide or signaling aptamer, thereby releasing the distance constraint between the biosensor electrode  206 ,  306 , or  406  and the respective redox reporter  204 ,  304 , or  404  on the signaling oligonucleotide or signaling aptamer, allowing a faradaic current to flow (ΔE). The magnitude increase in the electrochemical potential, measured in a handheld electrical meter, such as electrical meter  502  in  FIG. 5 , is directly proportional to the proteolytic activity in the biological sample. 
         [0055]    This change in the electrochemical potential (ΔE) is controlled by initially imposing distance constraints on the redox reporter  204 ,  304 , or  404 , which in the specific examples presented herein and shown in  FIGS. 2-4  is methylene blue (MB), and the distance constraints are released by hydrolysis of peptide substrate sequences by target proteases when they are present in the biological sample. Relative to optical biosensors, electrochemical biosensors are more cost effective, more stable, less susceptible to contaminants and non-specific binding, and involve the use of simpler instrumentations for detection. Furthermore, the detection of electrical signals is not affected by factors such as solution turbidity or opacity, which may cloud the detection of optical signals, including fluorescence. 
         [0056]    The first exemplary electrochemical biosensor configuration, depicted in  FIG. 2 , includes two entities: 1) a peptide-oligonucleotide conjugate (PepOliCon) and 2) a signaling oligonucleotide that binds to the oligonucleotide moiety of the PepOliCon via complementary DNA interactions. The PepOliCon  202  depicted in  FIG. 2  (a specific example of which is given by SEQ ID NO: 55) itself contains four interworking components that make the present invention work. First, a binding component  201 , such as sulfur, at its terminus used to bind or adhere the PepOliCon to the biosensor electrode  206 . Second, a lifting peptide sequence that elevates the redox reporter  204  on the adjacent signaling oligonucleotide  205  far enough above the biosensor electrode  206  to prevent current flow. Third, a protease cleavage sequence  203  that is cleaved by proteases present in the biological sample. Lastly, a DNA oligonucleotide  207  binds to a complementary DNA sequence on the signaling oligonucleotide  205 . 
         [0057]    Similarly, the signaling oligonucleotide  205  contains three interworking components that make the present invention work. First, a binding component  201 , such as sulfur, at one of its termini (5′ in the example given in SEQ ID NO: 56) is used to bind or adhere the signaling oligonucleotide  205  to the biosensor electrode  206 . Second, a sequence that is complementary to the DNA oligonucleotide sequence on the PepOliCon promotes a strong binding interaction between the PepOliCon  202  and the signaling oligonucleotide  205 . Third, a redox reporter  204 , such as methylene blue, attached to its other terminus (3′ in the example given in SEQ ID NO: 56). 
         [0058]    The second exemplary electrochemical biosensor  300  depicted in  FIG. 3 , using an aptamer system, works by a similar process to promote the flow of current once cleavage of the protease recognition sequence  303  by sample proteases has occurred.  FIG. 3  shows the aptamer configuration of the electrochemical biosensor of the present invention. This configuration also includes two entities: 1) an aptamer-recognition peptide construct  302  containing an aptamer epitope  307 , and 2) a signaling aptamer  305 . The aptamer recognition peptide construct  302  (a specific example of which is given by SEQ ID NO: 53) consists of four interworking components that make the present invention work. First, a binding component  301 , such as sulfur, at its terminus used to bind or adhere the aptamer recognition peptide construct to the biosensor electrode  306 . Second, a lifting peptide sequence that elevates the redox reporter  304  on the adjacent signaling aptamer  305  above the biosensor electrode  306  to prevent current flow. Third, a protease cleavage sequence  303  that, when cleaved by proteases present in the biological sample, releases the constraint of the lifting peptide that had been elevating the redox reporter  304  above the biosensor electrode  306  so as to prevent current from flowing. Fourth, a peptide aptamer epitope sequence  307  specifically recognized by an aptamer with which it forms a tight binding interaction. 
         [0059]    The signaling aptamer  305  contains four interworking components that make the present invention work. First, a binding component  301 , such as sulfur, at one of its termini (3′ in the example given in SEQ ID NO: 57) is used to bind or adhere the signaling aptamer  305  to the biosensor electrode  306 . Second, a spacer sequence is long enough to allow the peptide recognition sequence of the aptamer to reach up to the specific peptide sequence  307  on the aptamer recognition peptide construct  302  with which it forms a specific, high-affinity binding interaction. Third, the peptide recognition sequence makes a specific high-affinity binding interaction with the peptide aptamer epitope  307  on aptamer-recognition peptide construct  302 . Fourth, a redox reporter  304  such as methylene blue, attached to its other terminus (5′ in the example given in SEQ ID NO: 57). 
         [0060]    The peptide aptamer epitope/aptamer pair can either be chosen from a commercially available pair (e.g. aptamer APT00536 against a 6× His-tag; or APT00523 against a 15-mer acetylated peptide representing the N-terminal tail of Histone 450; or ATP00390 against amyloid peptide betaA4(1-40); or aptamers ATP00308, ATP00309, ATP00310, ATP00311, or ATP00312 against brain natriuretic peptide Ep1, Ep2, Ep3, EP4 or Ep5 (all available from Creative Biogene), or it can be derived from a unique peptide sequence and a new specific high affinity recognition DNA aptamer produced against it as a unique part of the present invention. 
         [0061]    The third exemplary electrochemical biosensor  400  depicted in  FIG. 4 , which uses an OligoPepOligo system, also works by a mechanism that promotes the flow of current once cleavage of a protease recognition sequence by proteases in a biological sample has occurred. This configuration also includes two entities: 1) an oligonucleotide-peptide-oligonucleotide (OligoPepOligo) conjugate  402 , containing a protease cleavage sequence  403 , and 2) a signaling oligonucleotide  405 . The OligoPepOligo  402  includes four interworking components that make the present invention work. First, a binding component  401 , such as sulfur, at one of its termini (3′ in the examples given in SEQ ID NOs: 60-61) is used to covalently bind or adhere the OligoPepOligo  402  to the biosensor electrode  406 . Second, a DNA sequence binds to a complementary DNA sequence on the signaling oligonucleotide, thereby forming a length of double stranded DNA that serves as part of the mechanism that elevates the redox reporter  404  far enough above the biosensor electrode  406  to prevent current flow. Third, a peptide sequence containing a protease cleavage site  403  is joined to oligonucleotide sequences at both its amino-terminal and carboxyl-terminal ends via highly efficient copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC), or strain-promoted alkyne-azide cycloaddition (SPAAC) “click” chemistry. When the peptide is cleaved by proteases in the biological sample, the lifting constraint of the double stranded DNA that holds the redox reporter  404  above the biosensor electrode  406  is released. Fourth, a second oligonucleotide sequence that binds to a complementary DNA sequence further along the signaling oligonucleotide  405 . 
         [0062]    The signaling oligonucleotide contains five interworking components that make the present invention work. First, a binding component  401 , such as sulfur, at one of its termini (5′ in the examples given in SEQ ID NOs: 58-59) is used to bind or adhere the signaling oligonucleotide to the biosensor electrode  406 . Second, a DNA sequence binds to a complementary DNA sequence on the OligoPepOligo, thereby forming a length of double stranded DNA that serves as the lifting mechanism that elevates the redox reporter  404  far enough above the biosensor electrode  406  to prevent current flow. Third, a short spacer such as the polythymine sequence seen in the center of both SEQ ID NOs: 58-59 which links the two oligonucleotide regions of the signaling oligonucleotide that each make binding interactions with complementary sequences on the OligoPepOligo. Fourth, the second oligonucleotide sequence that binds to a complementary sequence on the OligoPepOligo. Fifth, a redox reporter  404  such as methylene blue, attached to the other terminus of the signaling oligonucleotide (3′ in the examples given in SEQ ID NOs: 58-59). 
         [0063]    The exemplary OligoPepOligo electrochemical biosensor  400  depicted in  FIG. 4  can be configured such that only the signaling oligonucleotide is covalently attached to the biosensor electrode  406  while the OligoPepOligo does not contain a binding component  401  such as sulfur, but is attached to the biosensor only by its complementary DNA binding interactions to the signaling oligonucleotide. While those skilled in the art can appreciate that both configurations of making this biosensor are possible (i.e. the OligoPepOligo conjugate  402  may or may not contain a binding component  401 , such as sulfur), the fully covalent architecture significantly improves the stability of this OligoPepOligo biosensor and therefore provides a longer shelf life for a commercial product. 
         [0064]    Other important qualities of the present invention include, but are not limited to, the ability to provide accurate quantitation; to be free of interfering mechanisms that could lead to false results; and to have a large dynamic and linear range to be able to provide accurate measurement of wound protease activities that can be highly variable in different biological samples, even within chronic wound fluids. It also measures protease activity and not just protease concentration since proteases can be present in inactive forms in biological samples; is cost effective; easy to use; point-of-care; and has an intuitive readout with the detector signal readout from the electrical meter  502  increasing with increasing protease activity. 
         [0065]    Examples of human biological samples containing proteases for which the present invention is applicable include, but are not limited to: blood, lymph, wound fluid, saliva, gingival crevice fluid, nasal discharge, mucus from other membranes, bronchoalveolar lavage, urine, and lacrimal fluid. The present invention is also applicable to other bodily fluids in humans and animals and to biological fluids from other phyla such as the culture or growth media of microorganisms, from insects and other invertebrates, plants, and from water samples. One example of such a fluid would include the measurement of neutrophil elastase activity or other NSPs in gingival crevice fluid. Another example would be the measurement of neutrophil elastase activity or other NSPs in bronchoalveolar lavage. Another example would be the measurement of protease activity within wound fluid. In the embodiments exemplified herein, the present invention provides diagnostics for the detection and quantification of the activities of matrix metalloproteinases (MMPs) and neutrophil serine proteases (NSPs) at the site of a wound. 
         [0066]    The simplicity and utility of blood glucose monitors is well recognized as essential to the management of diet and insulin therapy in diabetics. The present invention is as simple and important in the management of the therapy of non-healing (chronic) wounds. Unlike the electrochemical sensing methods used in blood glucose measurement, which are based on solvent reorganization energies and which use redox reporters of ferrocene and osmium, the method of the present invention is based upon stereochemical distance constraints imposed by a lifting peptide or a lifting double-stranded DNA between a redox reporter  204 ,  304 , or  404 , such as methylene blue (MB) on the terminus of a signaling oligonucleotide or a signaling aptamer, and the surface of the biosensor electrode  206 ,  306 , or  406 . 
         [0067]    There are millions of patient visits annually to wound clinics throughout the United States, and globally. In an example embodiment of the present invention, the activity of MMPs will be determined. In another example embodiment, the activity of MMPs in wounds will be determined During the initial visit of each new patient presenting with a non-healing wound, use of the present invention indicates the level of protease activity in the wound at the point of care (POC), to guide the wound management strategy that is initially adopted for each new wound. The electrical meter  502  of the present invention is a single hand-held device shown in  FIG. 5  including a test strip either  600  or  700  shown in  FIGS. 6 and 7 , inserted into the detection input slot  501  of the hand-held electrical meter  502  that provides results in a digital readout of the wound protease activity. The electrical meter  502  and either test strip  600  or  700  of the present invention is simple for doctors, physician&#39;s assistants, nurses, patients, and other persons involved in the medical industry to use. 
         [0068]    The detection device  500  operates by inserting test strip  700 , for example, into the detection input slot  501  of electrical meter  502 , then drawing up a small volume (nanoliters to microliters) of biological sample  702  into the end of test strip  700  that is automatically controlled by capillary action into a sample channel  701  of set dimensions, and waiting approximately ten to fifteen minutes (10-15 min.) for the proteolytic chemistry to occur, after which the electrical meter  502  displays the level of protease activity on the screen. Electrical meter  502  senses whether the correct amount of biological sample  702  has been drawn into the sample channel slot  701  within the spacer  707 . Next, electrical meter  502  starts timing the reaction. At the end of the reaction time, approximately ten to fifteen minutes (˜10-15 min), which is preset in the detection device, the electrical meter  502  provides a numerical readout of the protease activity. 
         [0069]    In an example embodiment, the presence of active infection in a wound is assessed by inserting test strip  700 , for example, containing a biosensor for the measurement of NSPs, into a electrical meter  502  that is capable of measuring electrical potentials; drawing a small measured sample of wound fluid into the end of the test strip by capillary action; allowing an incubation time of approximately ten to fifteen minutes (˜10-15 min.) for proteolysis and electrochemical potential generation; and representing the proteolytic activity in the biological sample by a digital read-out on the electrical meter  502 . The number displayed on electrical meter  502 , quantitating the level of NSP protease activity in biological sample  702 , is entered into the patient&#39;s medical records by the wound care provider as an indication of the level of active infection. In another example embodiment, the activity of NSPs in gingival crevice fluid, using the same methodology, indicates the presence of plaque or tooth biofilm associated with gingivitis and/or periodontitis. In yet another example embodiment, the activity of NSPs in bronchoalveolar lavage indicates the level of protease activity in the lungs that are associated with active infections, irreversible lung tissue degeneration, such as in emphysema and other chronic obstructive pulmonary disease (COPD) in adults, and chronic lung disease (CLD) in infants. 
         [0070]    The present invention distinguishes itself from these other methods in that it uses a specific signal-on distance-constraining molecular architecture to control the stereochemistry of molecules attached to an electrode surface, such as the biosensor electrodes  206 ,  306 , or  406 , to ensure that an electrochemical potential is prevented or minimized prior to the introduction of biological sample. Specifically, the chemical constraint system utilizes lifting peptides or double-stranded DNA in peptide-oligonucleotide (PepOliCon) or oligonucleotide-peptide-oligonucleotide (OligoPepOligo) conjugates, respectively, or lifting peptides conjugated to aptamer epitopes to elevate a redox reporter  204 ,  304  or  404 , such as methylene blue, attached to a signaling oligonucleotide or a signaling aptamer, above the biosensor electrode  206 ,  306 , or  406 , until hydrolysis of specific protease cleavage sites incorporated into the biosensor construct by sample proteases. The distance constraints maintaining the redox reporter  204 ,  304  or  404  out of signal range from the biosensor electrode  206 ,  306  or  406 , respectively are released via enzyme hydrolysis by sample proteases, thereby allowing the redox reporter  204 ,  304 , or  404  to approach the biosensor electrode  206 ,  306  or  406 , respectively and initiate an electrochemical potential through the working electrode connector  605  or  708 , which potential is directly proportional to the protease activity present in a biological sample. 
         [0071]    Sequences 
         [0072]    There are peptide sequences, oligonucleotide sequences, and conjugates of both that are subjects of the present invention, as illustrated in the embodiments shown in  FIGS. 2, 3, and 4 . Preferred peptide sequences for the present invention are of three kinds: protease cleavage sequences; lifting peptide sequences that help elevate the redox reporter moiety above the electrode in the ready position; and third, either commercially available or unique peptide sequences that are aptamer antigens. 
         [0073]    Oligonucleotide sequences important to the present invention are also of three kinds: sequences that are conjugated to peptides (either PepOliCon or OligoPepOligo conjugates) and which are designed to be complementary to portions of sequences on signaling oligonucleotides; signaling oligonucleotides that support a redox reporter and/or a binding component, such as sulfur on their termini and contain complementary sequences to those conjugated to peptides; and specific aptamer sequences with recognition for specific peptides. The first two are deoxyribonucleic acid (DNA) sequences, whereas the third, aptamer-type sequences may also be constituted by ribonucleic acid (RNA) sequences. 
         [0074]    Protease Cleavage Sequences 
         [0075]    Proteases that contribute to the degradation of extracellular matrix proteins, cytokines, growth factors, and cell surface receptors in wounds include matrix metalloproteinses, MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, and MMP-13. Serine proteases also contribute to tissue degradation, often at sites of infection, and the present invention calls for Neutrophil Serine Proteases (NSPs) to be used as specific indicators of active infection. The primary serine proteases released from neutrophils that are recruited to sites of active infection and which indicate stress of the host are neutrophil elastase (NE), proteinase-3 (Pr3) and cathepsin-G (CG). Examples of substrate sequences used for the present invention include, but are not limited to those that are promiscuous for multiple proteases, or those that are specific, or somewhat discriminatory for individual proteases. 
         [0076]    Some examples of each are given below using the single letter designation code for amino acids. The cleavage position in each is indicated by a forward slash. 
         [0000]    
       
         
               
             
           
               
                   
               
             
             
               
                 PROMISCUOUS MMP SUBSTRATE SEQUENCES 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 SUBSTRATE SEQUENCES PROMISCUOUS FOR MMP-2 AND MMP-9 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 SUBSTRATE SEQUENCES MORE SPECIFIC FOR MMP-9 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 PROMISCUOUS NSP SUBSTRATE SEQUENCES 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 SUBSTRATE SEQUENCES MORE SPECIFIC FOR NE 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 Nle(O-Bzl) = 6-benzyl-norcleucine 
               
               
                 Met(O 2 ) = methionine dioxide 
               
               
                 Oic = octahydro-1H-indole-2-carboxylic acid 
               
               
                 Abu = aminobutyric acid 
               
               
                   
               
               
                 SUBSTRATE SEQUENCES PROMISCUOUS FOR MMPS AND NSPS 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
             
          
         
       
     
         [0077]    In the above sequences, the solid black line on the left of each represents a lifting peptide sequence (such as SEQ ID NOs: 48-50) or an oligonucleotide sequence that forms a double stranded DNA structure with a complementary sequence in another oligonucleotide construct (such as sequences contained within SEQ ID NOs: 58-59 form double-stranded DNA with complementary sequences contained within SEQ ID NOs: 60-61), either of which mechanism contributes to the elevation of the redox reporter above the biosensor electrode  206 ,  306 , or  406  surface in the ready configuration where the redox reporter  204 ,  304 , or  404 , respectively, cannot cause a faradaic current to flow through the biosensor electrode  206 ,  306 , or 406. In the above SEQ ID NOs: 1-47, the dashed black line on the right of each sequence represents oligonucleotide sequences (such as SEQ ID NO: 54), which are complementary to sequences in signaling oligonucleotides. The entire constructs are known as either peptide-oligonucleotide conjugate (PepOliCon) or oligonucleotide-peptide-oligonucleotide (OligoPepOligo) sequences. In the literature, peptide-oligonucleotide conjugate sequences are abbreviated as POC, but within the field of the present invention the abbreviation ‘POC’ is restricted to designate Point-of-Care, and herein peptide-oligonucleotide conjugate is abbreviated as PepOliCon. 
         [0078]    Examples of lifting peptide sequences include, but are not limited to the following. 
         [0079]    CEELRVRLASHLRKLRKRL (SEQ ID NO: 48). The de novo peptide structure prediction program known as PEP-FOLD, predicts that this peptide will adopt an a-helical structure that would be suitable as a lifting peptide in the present invention. Within its native apolipoprotein E structure this sequence also exists as an α-helix. The predicted elongated lifting structure of this peptide, with promiscuous MMP substrate (SEQ ID NO: 3) attached to its carboxyl-terminus (i.e. the sequence depicted as SEQ ID NO: 51) is indicated in  FIG. 1 . 
         [0080]    CVLVLVLVL (SEQ ID NO: 49). The de novo peptide structure prediction program known as PEP-FOLD predicts that this peptide will adopt an elongated structure that would be suitable as a lifting peptide in the present invention. 
         [0081]    DDAADDSADC (SEQ ID NO: 50). The de novo peptide structure prediction program known as PEP-FOLD predicts that this peptide will adopt an elongated structure that would be suitable as a lifting peptide in the present invention. 
         [0082]    An example of a peptide sequence that includes a lifting peptide with a protease substrate sequence attached at the terminus that would be elevated above the biosensor electrode  206  or  306  includes, but is not limited to the following. 
         [0083]    CEELRVRLASHLRKLRKRL-GPQG/IFGQ (SEQ ID NO: 51). The predicted structure of SEQ ID NO: 51, composed of lifting peptide SEQ ID NO: 48 with promiscuous MMP substrate SEQ ID NO: 3 joined at its carboxyl-terminus is shown in  FIG. 1 . 
         [0084]    CVLVLVLVL-GIATFC/MLMPEQ (SEQ ID NO: 52). This peptide is composed of lifting peptide SEQ ID NO: 49 with promiscuous NSP substrate SEQ ID NO: 37 joined at its carboxyl-terminus. 
         [0085]    An example of a polypeptide sequence consisting of a lifting peptide sequence, followed by a protease substrate sequence and then a specific aptamer recognition sequence (aptamer epitope; in this case the Histone 4-K16(Ac) N-terminal tail) includes, but is not limited to the following: 
         [0000]    
       
         
               
             
           
               
                 OLIGONUCLEOTIDE SEQUENCES 
               
               
                 (SEQ ID NO: 53) 
               
               
                 CEELRVRLASHLRKLRKRL-GPQG/IFGQ-GGKGLGKGGAK(Ac)RHRK  
               
             
          
         
       
     
         [0086]    Examples of DNA oligonucleotide sequences include, but are not limited to, the following. 
         [0087]    (a) DNA sequence that binds to a complementary DNA sequence in another oligonucleotide sequence and which can be conjugated to lifting peptides in PepOliCons, or peptides constituting protease cleavage sites in OligoPepOligos. 
         [0000]    
       
         
               
               
             
           
               
                   
                 (SEQ ID NO: 54) 
               
               
                   
                 5′-CCATCTCCACTGC-3′  
               
             
          
         
       
     
         [0088]    (b) example of a fully conjugated peptide-oligonucleotide (PepOliCon). [For clarity, in the PepOliCon and OligoPepOligo conjugate sequences, amino acids are depicted using their conventional three-letter code and nucleic acids are depicted using their conventional single-letter codes]. 
         [0000]    
       
         
               
             
           
               
                 (SEQ ID NO: 55) 
               
               
                 Cys-Glu-Glu-Leu-Arg-Val-Arg-Leu-Ala-Ser-His-Leu- 
               
               
                 Arg-Lys-Leu-Arg-Lys-Arg-Leu-Gly-Pro-Gln-Gly/Ile- 
               
               
                 Phe-Gly-Gln-Lys(N 3 )-5′hexynyl-CCATCTCCACTGC-3′  
               
             
          
         
       
     
         [0089]    (c) signaling oligonucleotide to be used in the PepOliCon Biosensor configuration. 
         [0000]    
       
         
               
             
           
               
                 (SEQ ID NO: 56) 
               
               
                 3′-MB-(CH 2 ) 2 -C-GGTAGAGGTGACG-TTGGGTGTTGTGTCCAACCC- 
               
               
                 (CH 2 ) 6 -S-S-(CH 2 ) 6 -5′ 
               
             
          
         
       
     
         [0090]    (d) signaling oligonucleotide aptamer sequence (includes H4-K16(Ac) N-terminal tail aptamer) 
         [0000]    
       
         
               
             
           
               
                 (SEQ ID NO: 57) 
               
               
                 5′-MB-(CH 2 ) 2 -C-GTAAGTTAATTGGACTTGGTCGTGTGCGGCACAGC 
               
               
                 GAT-TTGGGTGTTGTGTCCAACCC-(CH 2 ) 6 -S-S-(CH 2 ) 6 -3′  
               
             
          
         
       
     
         [0091]    (e) signaling oligonucleotides to be used in the OligoPepOligo Biosensor configuration. 
         [0000]    
       
         
               
             
           
               
                 (SEQ ID NO: 58) 
               
               
                 5′-SH-(CH 2 ) 6 -CCATCTCCACTC-GTTTTT-CGTGCTTCTG- 
               
               
                 (CH 2 ) 2 -MB-3′  
               
               
                   
               
               
                 (SEQ ID NO: 59) 
               
               
                 5′-SH-(CH 2 ) 6 -CGTGCTTCTGCT-GTTTTT-CCATCTCCAC- 
               
               
                 (CH 2 ) 2 -MB-3′  
               
             
          
         
       
     
         [0092]    (f) oligonucleotide-peptide-oligonucleotide (OligoPepOligo) sequences 
         [0000]    
       
         
               
             
           
               
                 (SEQ ID NO: 60) 
               
               
                 3′-SH-(CH 2 ) 6 -GGTAGAGGTGAG-TriazoleLinker1-Gly- 
               
               
                 Pro-Gln-Gly/Ile-Phe-Gly-Gln-TriazolLinker2- 
               
               
                 GCACGAAGAC-T-5′  
               
               
                   
               
               
                 (SEQ ID NO: 61) 
               
               
                 3′-SH-(CH 2 ) 6 -GCACGAAGACGA-TriazoleLinker1-Ala- 
               
               
                 Ala-Pro-Val/Leu-Ser-Ala-Gln-TriazoleLinker2- 
               
               
                 GGTAGAGGTG-T-5′ 
               
             
          
         
       
     
         [0093]    Peptide-oligonucleotide (PepOliCon) and oligonucleotide-peptide-oligonucleotide (OligoPepOligo) conjugates can be produced by the highly efficient copper(I)-catalyzed alkyne-azide cyloaddition (CuAAC) or Strain-Promoted Alkyne-Azide Cycloaddition (SPAAC) “click” chemistry. CuAAC conjugation is efficient for azido-modification of amines at either the 3′-end or the 5′-end of an oligonucleotide. A second oligonucleotide modification approach, specific to the 5′-terminus consists of adding 5′-bromohexyl phosphoramidite in the last synthesis cycle. This modifier can then be easily transformed into a 5′-azido group by displacement of bromide using sodium azide. The first method can be performed in solution while the other allows the azide addition to be performed in solid phase on the synthesis column prior to cleavage and deprotection of the oligonucleotide. If the oligonucleotide is derivatized with azide, the peptide moiety is derivatized with alkyne, for example by alkyne-activated ester. Peptides can also be derivatized at either the amino- or carboxyl-terminal end by azide, and in that case alkyne-containing oligonucleotide can be prepared with alkyne phosphoramidite. 
         [0094]    Copper-free “click” chemistry is also available. In this case, the oligonucleotide entity is terminally labeled with a cyclooctyne compound (such as DBCO or BCN) and the peptide binding partner is labeled with azide. The reaction of these partners is known as strain-promoted alkyne-azide cycloaddition (SPAAC), and results in a cross-linked triazole conjugate. Diarylcyclooctynes are thermostabile with very narrow and specific reactivity towards azides, resulting in almost quantitative yields of stable triazoles. The use of the strained cyclooctyne decreases the activation energy for the cycloaddition “click” reaction, enabling it to be carried out without the need for catalysis at low temperatures with an efficiency greater than that of the Cu(I)-catalyzed ligation. In another rendition of the “click” chemistry, conjugation can also be effected by inclusion of a cysteine at the peptide terminus to be conjugated using succinimidyl ester linkage, such as SMCC with a C6 amino linkage that binds to the oligonucleotide terminus. 
         [0095]    Thus, it is seen that the objects of the present invention are efficiently obtained, although modifications and changes to the invention should be readily apparent to those having ordinary skill in the art, which modifications are intended to be within the spirit and scope of the invention as claimed. It also is understood that the foregoing description is illustrative of the present invention and should not be considered as limiting. Therefore, other embodiments of the present invention are possible without departing from the spirit and scope of the present invention.