Patent Publication Number: US-2017356913-A1

Title: Method for Detecting Human Butyrylcholinesterase

Description:
The invention relates to the field of detection of biological compounds, particularly for the detection of butyrylcholinesterase. Such a detection may be advantageously used in the detection of the duration and severity of exposure to cholinesterase inhibiting nerve gases or pesticides. 
     INTRODUCTION 
     Organophosphorous compounds are a group of the most toxic compounds that are known. Many of those have been used in warfare as nerve gas, such as sarin, tabun and VX. Organophosphorous toxins are also used as pesticides and insecticides. 
     Intoxication with these compounds brings a serious risk. The potential for civilian exposure to these compounds is greater today than at any time in history. Several events in recent history, such as the gassing of Kurds in Iraq in 1988, the attack in the subway in Tokyo in 1995 and the recent still debated gas attacks in Syria, indicate a demand for more accurate and sensitive methods to monitor exposure to cholinesterase inhibiting agents. 
     Currently one of the most reliable methods to measure exposure to organophosphorous compounds is by using inhibition of bytyrylcholinesterase (BuChE or BChE) as an indicator. Whereas originally such an assay was performed based on a mass spectrographic detection (Polhuijs, M. et al., 1997, Toxicol. Appl. Pharmacol. 146:156-161), more recently MALDI-TOF methods have been used (e.g. Doom, J. A. et al., 2001, Toxicol. Appl. Pharmacol. 176:73-80; Fidder, A. et al., 2002, Chem. Res. Toxicol. 15:582-590; Sun, J. and Lynn B. C., 2007, J. Am. Soc. Mass Spectrom. 18:698-706) and also other enzymatic assays have been reported (Du, D. et al., 2011, Anal. Chem. 83:3770-3777; Du, D. et al., 2012, Anal. Chem. 84:1380-1385). 
     Although the mass spectrometric assays have been found to perform very well in measuring exposure to organophosphorous compounds, the equipment needed is large and heavy and can therefore not being used in field situations. Further, the mass spectrometric assay requires a relatively long analysis time (typically about 4 hours), due to the sample work-up that is needed, as well as highly trained personnel. Therefore, there is a need for a fast, reliable and low weight device for performing the assay in the field. 
     SUMMARY OF THE INVENTION 
     The inventors now have developed an optical resonator biosensor for the detection of butyrylcholinesterase comprising two optical resonator biosensor systems in which in the first optical resonator biosensor system a probe is attached to the resonator wherein said probe is able to bind butyrylcholinesterase, preferably wherein said probe is able to bind uninhibited butyrylcholinesterase. In a further preferred embodiment the optical resonator biosensor provides in the second optical resonator biosensor system an antibody that is attached to the resonator, wherein said antibody is able to bind butyrylcholinesterase, preferably wherein said antibody is able to bind both inhibited and uninhibited butyrylcholinesterase. 
     In a further preferred embodiment the optical resonator biosensor is provide with a sample, applied in such a way that it comes into contact with both optical resonator biosensor systems, preferably by applying the the sample via a capillary track. In a further preferred embodiment, both optical resonator biosensor systems are placed along one capillary. In such a system preferably the amount of probes in said first optical resonator biosensor is sufficient large to capture all the uninhibited butyrylcholinesterase. Alternatively, also preferred is a biosensor wherein one optical resonator biosensor system is placed along one branch of the capillary, while the other optical resonator biosensor system is placed along an other branch of the capillary. 
     A further preferred embodiment is a biosensor wherein the resonator is a ring resonator. 
     Another further preferred embodiment is a biosensor wherein the resonator biosensor is a waveguide, preferably wherein said waveguide is part of an interferometer. 
     A further part of the invention is a method for determining the exposure to an organophosphorous compound by measuring the amounts of inhibited, uninhibited and total butyrylcholinesterase in a sample by assaying said sample with an optical resonator biosensor according to the invention. 
     Also part of the invention is the use of an optical resonator biosensor for the determination of the exposure to an organophosphorous compound, wherein said optical resonator biosensor is an optical resonator biosensor according to the invention. 
     In the above mentioned optical resonator biosensor, method and use the butyrylcholinesterase is a human butyrylcholinesterase. Further, in the above mentioned optical resonator biosensor, method and use the probe comprises the following compound: 
     
       
         
         
             
             
         
       
     
     in which R is an spacer chain with more than 10 C-atoms, which is able to be bound to the sensor surface. Preferably said spacer chain is a substituted or unsubstituted, linear or branched hydrocarbon chain of more than 10 carbon atoms, wherein one to three of the C atoms may be replaced by a heteroatom selected from the group of O, N and S, and wherein the chain may have substitutions selected from the group of halogen, phenyl, linear or branched O—C 1 -C 6  alkyl, linear or branched alkoxy, nitro, cyano or methylsulfinyl. Examples of such probes are: 
     
       
         
         
             
             
         
       
     
     In a further preferred embodiment the antibody that binds to butyrylcholinesterase in the above mentioned optical resonator biosensor, method and use is monoclonal antibody 3E8. 
    
    
     
       DESCRIPTION OF THE FIGURE 
         FIG. 1  shows the dose-dependent shift of resonsnace wavelength from various doses of human butyrylcholinesterase applied to a biosensor on a ring resonator. The Y-axis denots the shift in wavelength, while the x-axis shows the time in seconds. 
     
    
    
     DETAILED DESCRIPTION 
     Butyrylcholinesterase is an enzyme that is inhibited when an organosphosphorous compound, such as a nerve gas or a pesticide, binds to its active site. Inhibition of BuChE in itself does not give any toxic effects (in comparison to inhibition of acetylcholinesterase, AChE, which causes the toxicity demonstrated by the organophosphorous compounds), but it can ideally be used as a biomarker. The concentration of inhibited BuChE in comparison with the total BuChE concentration conveys information whether or not a subject has been exposed to an organophosphorous compound, as well as an indication of the extent of the exposure. 
     Most systems use detection of inhibited BuChE and indeed this indicates exposure more qualitatively. For a quantitative assay, however, preferably also detection of total BuChE is achieved with immunological methods. 
     To detect total BuChE preferably an antibody that is able to bind to the enzyme is used. To detect the amount of inhibited BuChE it is easier to achieve this by detecting the amount of uninhibited BuChE, which can be done by detection of binding to a probe that is able to bind uninhibited BuChE. Of course then the amount of inhibited BuChE is the total amount of BuChE minus the amount of uninhibited BuChE. As used in the present application, the word “probe” is used for a molecule that is able to bind uninhibited BuChE. 
     As used in the present application, the term ‘inhibited BuChE’ means an enzyme that is inactivated by binding to a target molecule, said target molecule being an organophosporous compound as provided by a nerve gas or pesticide, or, alternatively, provided as probe in the assay of the present invention. Accordingly, an ‘uninhibited BuChE’ of the present invention is a molecule wherein the recognition site for an organophosphorous compound has not been occupied, which in general means that the enzyme is still able to exert its enzymatic action. Further, the ‘inhibited’ or ‘uninhibited’ state is not altered by binding to the ant-BuChE antibody as defined herein. 
     As used herein an anti-BuChE antibody, often being referred to as ‘the antibody’ in the present description, is preferably a monoclonal antibody that is able to bind to both inhibited and uninhibited BuChE. Such antibodies are known in the art (e.g. Wang, L. et al., 2011, Analytica Chimica Acta, 693(5):1-6; Sproty, J. L., et al. 2010, Anal. Chem. 82:6593-6600). One example is monoclonal antibody 3E8. An alternative antibody might be the goat polyclonal IgG N-15 Sc-46803 (against N-terminal part of human BuChE; Santa Cruz Biotechnology). 
     In the present invention the detection of the total and uninhibited BuChE is achieved by using an optical detection method, an optical resonator biosensor or ring resonator. An optical resonator biosensor works through monitoring resonance wavelength shifts which occur in optical response of the ring resonator, i.e. its transmission as function of wavelength. The binding of the biomolecule to another molecule (a probe, or an antibody) or of a biomolecule-antibody or biomolecule-probe complex to the sensor will result in a shift of resonance wavelengths allowing detection of bound vs unbound biomolecule. 
     An optical resonator biosensor according to the present invention consists of three parts: 1) a biorecognition element, which in the present invention is a probe or an antibody that is able to bind BuChE, more preferably human BuChE; 2) an optical transducer (the optical resonator) and 3) an electronic readout scheme for measuring and recording optical frequency shifts (Vollmer, F. and Baaske, M. 2012, ChemPhysChem 13:427-436). The optical resonator may be in the form of a glass microsphere, but may also be formed by micro-capillaries and liquid-core optical ring resonators (LCORR), micro-toroids, micro-disks, photonic crystal cavities and micro-rings. Preferably in the present invention a ring resonator is used. The general lay-out of such micro-rings is known in the art (e.g. Lin, S. Y. et al., 2010, Nano Lett. 10:2408-2411; Nitkowski A. et al., 2011, Biomed. Optics Express 2:271-277; Carlborg, C. F. et al., 2010, Lab on a Chip 10:281-290). 
     The advantages of the optical resonator biosensor are that a complete test system can be obtained on a very small scale: the actual measuring device can be provided as a lab-on-a-chip technology, where e.g. a ring resonator maybe used with a diameter of 5-100 μm. This allows testing in the field. 
     Further, the speed of detection can be very high (within a few seconds to a few minutes), while the amount of material needed, the sample volume, may be minimal (in the nanoliter or microliter range). On top of all of these benefits, the optical resonator biosensor systems usually combine a very high sensitivity with a good specificity. 
     The function of a ring resonator is characterized by a transmission as function of wavelength that is dependent on the ambient refractive index of the ring. This response shows periodic resonances. By monitoring the resonances wavelength shifts as function of the time, refractive index variations can accurately and immediately be measured. As such, the binding of molecules to its receptors (such as binding of the probe or antibody to the enzyme) will result in a shift of resonance wavelengths, the amount of shift corresponding to the amount of molecules bound. 
     A sample may be obtained from any body fluid that contains the enzyme butyrylcholinesterase, but preferably a blood sample. Since only a small amount of sample is needed to be applied to the testing device, blood from a finger prick is an ideal source, since it is readily obtained even under field conditions. However, also blood samples from other sources (e.g. venipuncture, or bleeding wounds) may be used. 
     The optical resonator biosensor in the present invention at least needs to measure the uninhibited BuChE level. For this purpose the resonator is provided with a probe that is able to bind uninhibited BuChe, more preferably uninhibited human BuChE (HuBuChE). This probe advantageously is a compound that is able to bind to the BuChe at the active site, i.e. a compound that resembles an organophosphorous compound as defined above or any other molecule that is able to bind to the active site of the enzyme. Preferably, this is a compound with the general formula: 
     
       
         
         
             
             
         
       
     
     in which R is an spacer chain with more than 10 C-atoms, which is able to be bound to the sensor surface. Preferably said spacer chain is a substituted or unsubstituted, linear or branched hydrocarbon chain of more than 10 carbon atoms, wherein one to three of the C atoms may be replaced by a heteroatom selected from the group of O, N and S, and wherein the chain may have substitutions selected from the group of halogen, phenyl, linear or branched O—C 1 -C 6  alkyl, linear or branched alkoxy, nitro, cyano or methylsulfinyl. Alternatively, the chain may comprise a (poly)ethylene glycol chain. Preferably the part of R that is capable of binding to the sensor is a biotin moiety or an amino moiety. In the fall of a biotin moiety, the sensor binds with an avidin moiety at the surface of the sensor. In the fall of an amino moiety, said moiety binds with a reactive carboxyl-group that is present at the surface of the biosensor. The skilled person will know how to provide the biosensor surface with the above-mentioned components for binding with their counterparts on the probe.
 
Examples of such probes are:
 
     
       
         
         
             
             
         
       
     
     It will be clear to the skilled person that the R group may vary considerably and primarily acts as a spacer to prevent interaction of the bound enzyme with the biosensor. 
     Next to the embodiments where the probe is bound to the surface of the sensor, it is also possible that an aqueous composition comprising free probe is added to the sample on the sensor. In such a case, the probe may first bind with the enzyme and then together with the enzyme may be bound to the sensor surface. Whether or not the actual binding to the enzyme in such a situation takes place before or after the probe has attached itself to the sensor surface is not relevant. 
     When such a molecule is immobilized on a refractive index sensor, such as a ring resonator, the binding of the molecule by BuChE or HuBuChE or the binding of the enzyme-antibody or enzyme-probe complex, can be detected as refractive index variations by monitoring the shift of the resonance wavelength as a function of time. Since the amount of shift corresponds to the amount of molecules bound, this provides a reliable, quick and a quantitative detection of the amount of uninhibited BuChE. 
     In one embodiment of the present invention, a test system comprises at least two sensors, one for measuring the amount of uninhibited BuChE, as indicated above, and one that measures the amount of inhibited BuChE. This latter measurement is achieved by measuring the total amount of BuChE, from which the amount of uninhibited BuChE then again should be subtracted (inhibited BuChE+uninhibited BuChE=total BuChE). As indicated above, this is achieved by measuring the binding of BuChE to an antibody, wherein said antibody is capable of binding both inhibited and uninhibited BuChE. The antibody may be bound to the surface of the sensor or may first react freely in the test (sample) solution with the BuChE and after reaction as antibody-enzyme complex be bound to the sensor. The skilled person is capable of finding ways how to bind the antibody to the sensor (e.g. by immobilising on the sensor an antibody that recognizes the anti-BuChe-antibody). An example of an antibody that is able to bind to both inhibited BuChE and uninhibited BuChE may be the monoclonal antibody 3E8, which is commercially available (e.g. from Thermo Fisher Scientific, Inc, Rockford, Ill., USA). Also other commercially available antibodies which are specific for (human) BuChE maybe used. 
     In this embodiment a sample is provided to a sample inlet after which the sample flows in two capillaries each to a different sensor. The signals obtained from those sensors are processed to calculate the concentrations of uninhibited and total BuChE. This enables calculation of the percentage inhibited BuChE and as such an indication of the exposure to organophosphorous compounds. 
     In another embodiment the two sensors are placed along one and the same capillary that runs from the sample inlet site. In this embodiment the sample flows across one sensor to measure the concentration of one analyte, and the same sample subsequently flows across the second sensor to measure the other analyte. Of course in this embodiment the probe and/or the antibody may be available as free soluble in the sample solution or bound to the sensor surface. Again, the signals from both sensors are then further processed to ultimately yield the concentration and/or percentage of inhibited BuChE in the sample. Such a sequential arrangement is allowed when the first sensor only binds a small fraction of the analyte (not depeleting the sample) such that the total concentration of analyte in the sample is not markedly affected. 
     In a further embodiment, the total amount of BuChE is measured by one sensor in one branch of the capillary, while in a second branch of the capillary all uninhibited BuChE is captured by a gel in which an excess amount of probe is available. After capturing all the uninhibited BuChE from the sample, the remaining amount of BuChE, which then necessarily is inhibited BuChE, is measured with immobilized antibodies. In the same embodiment the first measurement in the first capillary may also be a measurement of the amount of uninhibited BuChE, since the total amount of BuChE, and thus the percentage of inhibited BuChE, can be calculated in either way. In a very simplified form of this embodiment, the amount of uninhibited BuChE is measured quantitatively, while still depleting the sample from this uninhibited BuChE. This can be accomplished by having an excess amount of probe on the sensor. Since, beforehand it is not known how high the concentration of uninhibited BuChE will be, it would be possible to accommodate a dilution series of the sample to be measured by a series of sensors, where the sample is split and diluted in such a way that there is at least one sensor which would contain an excess amount of probe in order to completely delete this (diluted) sample. 
     According to the above description, the analysis device may be a lab-on-a-chip, which has a sample inlet site at which a sample to be assayed, such as a drop of blood, is applied. Optionally, a further aqueous solution containing a free soluble probe or antibody as described above may be added. Further, this ‘chip’ has one, two or more capillaries in which the sample is further guided to the one, two or more sensors at which the analyte is eventually detected. The signals that are obtained from these, one, two or more sensors are being sent to a calculating unit, which calculating unit may be a micro-computer, which is situated on the chip itself, or the calculating unit may be removed from the sensors and the results are transmitted. Temporarily, the signals can be stored in a storage medium on the chip and can be read out when contacting the chip with a transmission unit or directly with a calculating unit. Contacting the chip with a transmission device or calculating device may be in the form of any kind of contact that is able to transfer the signals, such as cables, a bus-like structure, such as a USB-stick or the like. Alternatively, the signals may be transmitted directly by a wireless transmitter, which may be any kind of wireless transmission system, i.e. modulated or unmodulated, encrypted or non-encrypted, radiosignals, optic, sonic or electromagnetic, etc. Preferably such a wireless transmission system is a WiFi system or an infra-red system. 
     Preferably, the biosensor system also provides an indicator which generates a signal when an excess amount of inhibited BuChE has been detected. Such an indicator can provide a chemical, electrical, optical or acoustical signal. The signal may be directly produced by one or more of the reagents contained in the system, but it may also be produced indirectly through input from the calculating unit. 
     As will be appreciated by one skilled in the art, aspects of the present invention may be embodied as a system, method or computer program product. Accordingly, aspects of the present invention may take the form of an entirely hardware embodiment, or an embodiment combining software and hardware aspects that may all generally be referred to herein as a “circuit,” “module” or “system.” Furthermore, aspects of the present invention may take the form of a computer program product embodied in one or more computer readable medium(s) having computer readable program code embodied thereon. Program code embodied on a computer readable medium may be transmitted using any appropriate medium, including, but not limited to, wireless, wireline, optical fiber cable, RF, etc., or any suitable combination of the foregoing. 
     Example 
     An antibody against human butyrylcholinesterase was immobilized on the surface of an integrated optical chip via streptavidin-biotin interactions. Initially, biotin was covalently bound to the chip. Subsequently, streptavidin (40 ug/ml in phosphate buffer saline) was applied and bound to the chip-immobilized biotin and finally a solution with biotinylated antibodies (3E8, 1 ug/ml in phosphate buffer saline) was applied such that the antibody was able to bind to the streptavidin. In order to apply the solutions in a well-controlled manner, a perspex flow cell was adhered to the optical chip with a UV-curing adhesive, such that a well-defined flow channel (W×h=3 mm×0.5 mm) above the surface of the optical chips is obtained. After this process the chip is ready to detect binding of human butyrylcholinesterase. 
     The integrated optical chip carries a ring resonator. The ring resonator has a length of 500 um. During the experiments the shift of the resonances in the optical response (transmission of the ring resonator) was measured. To this end, a tunable laser was continuously swept in steps of 1 pm across 4 nm and for each sweep the transmission of the ring was recorded as function of applied wavelength. From the recorded spectra over time, the shift of the resonance wavelengths as result of refractive index changes caused by human butyrylcholinesterase binding to the antibodies, is determined and plotted as function of time. This experiment was performed for different concentrations of human butyrylcholinesterase in phosphate buffer saline, that were applied to the sensor at a flow rate of 30 ul/min. As is shown in  FIG. 1  a dose-dependent shift in resonance wavelength could be observed.