System and method for voltage measurements on biological tissues

Abstract

The present invention relates to a system and method useful for determining the voltage of biological tissues and therefore to detect whether such tissues are cancerous.

Claims

1. A method for detecting cancerous tissue, comprising: contacting a sample of the tissue with a system for measuring the voltage of a tissue comprising: a tungsten electrode; and a silver/silver chloride electrode; detecting the voltage of the tissue sample; and comparing the voltage of the tissue sample to the voltage of a control sample.

2. The method according to claim 1, wherein a decreased voltage of the tissue sample compared to the control sample is indicative of cancer.

3. The method according to claim 1, wherein the tissue sample is in contact with the tungsten electrode and a medium is present that is in contact with the tissue and the silver/silver chloride electrode.

4. The method according to claim 1, wherein the tissue sample is in contact with the tungsten electrode and the silver/silver chloride electrode and a medium is present that is in contact with the silver/silver chloride electrode.

5. The method according to claim 1, wherein the silver/silver chloride electrode is a double junction reference electrode.

6. The method according to claim 1, wherein the system further comprises an instrumentation amplifier.

7. The method according to claim 1, wherein the system further comprises a medium.

8. The method according to claim 7, wherein the silver/silver chloride electrode is present within a housing, and the medium is in contact with at least the housing of the silver/silver chloride electrode.

9. The method according to claim 7, wherein the medium is also in contact with the tissue whose voltage is being measured using the system.

10. The method according to claim 9, wherein the medium is a cell culture medium.

11. The method according to claim 1, wherein one or both of the tungsten electrode and the silver/silver chloride reference electrode are held in place using a pipette tip.

12. The method according to claim 11, wherein the pipette tip has a plug.

13. The method according to claim 1, wherein the system is incorporated into a portable device.

14. The method according to claim 13, wherein the portable device further comprises a printed circuit board (PCB).

15. The method according to claim 14, wherein the portable device is connected to a data acquisition system.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The present invention will now be further described by way of reference to the following Examples which are present for the purposes of illustration only. In the Examples, reference is made to a number of Figures in which:

(2) FIG. 1 is a schematic of one arrangement of the system of the invention, in which the housing of the silver/silver chloride reference electrode and the tissue sample is in contact with a medium. This is referred to herein as the experimental setup with medium. In this Figure and also in FIGS. 7 and 10, the IA polarity [the plus (+) and minus () inputs of the IA] is indicative only.

(3) FIG. 2 is a simplified DC equivalent of the setup shown in FIG. 1.

(4) FIG. 3 shows the variation of measured potential difference with tungsten electrode tip depth (media only measurement).

(5) FIG. 4 shows the results of experiments to determine the voltage of medium alone, cancerous and non-cancerous tissue.

(6) FIG. 5 is a box-and-whisker plot depicting the quartiles of the voltage values shown in FIG. 3.

(7) FIG. 6 shows the difference in voltage values between the non-cancerous and cancerous omentum in individual patients.

(8) FIG. 7 is a schematic of another arrangement of the system of the invention, in which only the housing of the silver/silver chloride electrode is in contact with a medium. This is referred to herein as the experimental setup without medium.

(9) FIG. 8 shows the results of experiments to determine the voltage of cancerous and non-cancerous tissue using the experimental setup without medium.

(10) FIG. 9 shows the results of experiments to determine the voltage of medium alone, non-cancerous omental tissue and various different types of cancerous tissue using the experimental setup with medium.

(11) FIG. 10 is a schematic of the experimental setup used for experiments to determine the voltage of gels characterised by the same densities but different ion contents and gels characterised by the same ion contents but different densities. This experimental setup is very similar to the one shown in FIG. 1, except that the gel replaces the tissue whose voltage is to be determined and that there is no pipette holding the tungsten electrode in place.

(12) FIG. 11 shows the results of experiments to determine the voltage of gels characterised by the same densities but different ion contents (same agar concentration but different medium concentration).

(13) FIG. 12 shows the results of experiments to determine the voltage of gels characterised by the same ion contents but different densities (same medium concentration but different agar concentration).

(14) FIG. 13 is a detailed view of the electrodes that are part of a device in accordance with the invention. The first tungsten electrode measures biopotential and the second tungsten electrode transfers the printed circuit board's ground to the tissue. The third electrode is an Ag/AgCl double junction reference electrode.

(15) FIG. 14 is a detailed view of the Ag/AgCl double junction reference electrode that is part of a device in accordance with the invention. The double junction reference electrode is formed by inserting an Ag/AgCl reference electrode in a special chamber that contains cell culture medium.

(16) FIG. 15 is a schematic of a device in accordance with the invention, which also includes a printed circuit board (PCB) that is present in the interior of the device and a wireless connection to a computer. As can be seen from the Figure, the three electrodes that are located in the front side/tip of the device come in direct contact with the tissue when in use.

(17) FIG. 16 is a schematic of a tungsten electrode that was part of the experimental setups used in the Examples. The impedance of a typical electrode used was measured at 1000 Hz with a maximum current of 10 nA, at a tip immersion depth of 1 mm in saline solution. The value of the electrode impedance is 30.6 M and its metal-tip diameter is 0.3 mm.

(18) The dimensions illustrated in the Figures are exemplary only. The Figures are not drawn to scale. It will be appreciated that the dimensions and materials of the system of the invention can be varied as desired.

EXAMPLES

Example 1Measuring the Voltage of Paired Cancerous and Non-Cancerous Tissue Using Experimental Setup With Medium

(19) Experimental Setup

(20) A. Electrode Surface Potential Considerations

(21) FIG. 1 illustrates the experimental setup used for the recording of tissue voltage. It incorporates an instrumentation amplifier (IA) whose input terminals are connected to: i) a Ag/AgCl electrode in contact with the media within which the cancerous or non-cancerous omentum is touching, and ii) an FHC D.ZAP tungsten electrode with a metal-tip diameter of 0.3 mm. The tungsten electrode is in contact with the tissue sample which is placed within a standard 1 ml pipette tip placed in a beaker containing media; the electrode is not in direct contact with the media in the beaker. The Ag/AgCl electrode realises a high-impedance liquid junction path. The IA records and amplifies potential differences between its terminals when the system is in equilibrium. The amplified potential difference is subsequently converted to the digital domain by means of a data acquisition system.

(22) FIG. 2 illustrates a simplified DC equivalent of the setup shown in FIG. 1 which in practice records the difference between the potential developed on the surface of the tungsten electrode (when in contact with the tissue) and the Ag/AgCl electrode. During measurement the media corresponds to electrical ground. The double-layer capacitance of the tungsten-electrolyte (media) equivalent is denoted by C.sub.dl, its charge transfer by R.sub.ct while its solution resistance is denoted by R.sub.s. Bearing in mind that the recorded potential difference is practically of a DC nature, the IA equivalent circuit degenerates into two input capacitors, C.sub.IA.sup.+ and C.sub.IA.sup., associated with the IA's respective input terminals. When tissue voltage measurement takes place, a surface potential value is associated with a total charge
Q.sub.tot=A=AC.sub.dl+C.sub.IA.sup.+(1)

(23) developed across the capacitance C.sub.IA.sup.+ and AC.sub.dl where C.sub.dl denotes double-layer capacitance per unit area, A is the electrode-electrolyte interface area and denotes the surface charge density at the interface. However C.sub.dl can be determined as (W. Franks et al., Biomedical Engineering, IEEE Transactions on, vol. 52, no. 7, pp. 1295-1302, 2005 and M. R. Abidian and D. C. Martin, Biomaterials, vol. 29, no. 9, pp. 1273-1283, 2008):

(24) $\begin{array}{cc}\frac{1}{C_{\mathrm{dl}}^{}}=\frac{1}{C_H}+\frac{1}{C_G}=\frac{d_{\mathrm{OHP}}}{{\mathrm{.Math.}}_0{\mathrm{.Math.}}_r}+\frac{L_D}{{\mathrm{.Math.}}_0{\mathrm{.Math.}}_r\cosh \left(\frac{z}{2U_t}\right)}=\frac{1}{}& \left(2\right)\end{array}$

(25) where d.sub.OHP denotes the double-layer capacitor thickness, .sub.0.sub.r denotes the electrolyte's relative permittivity, L.sub.D denotes the Debye length, z denotes ionic chemical valence in the electrolyte and U.sub.t denotes the thermal voltage. Considering (1) and (2) yields:

(26) $\begin{array}{cc}=\frac{}{+\frac{C_{\mathrm{IA}}^{+}}{A}}& \left(3\right)\end{array}$
Bearing in mind (2), note that the quantity appears on both sides of the transcendental equation (3). It should be stressed that the derivation of the DC equivalent of the setup shown in FIG. 1 relies upon the strong assumption that the electrode-(electrolyte plus tissue) interface (see FIG. 1) can be described, at least to a first order, by the Gouy (R. Reeves, The electrical double layer: The current status of data and models, with particular emphasis on the solvent, in Modern Aspects of Electrochemistry. Springer, 1974, pp. 239-368)-Chapman (D. L. Chapman, The London, Edinburgh, and Dublin Philosophical Magazine and Journal of Science, vol. 25, no. 148, pp. 475-481, 1913) double-layer theoretical approach resulting from the combination of Poisson equation of electrostatics and Boltzmann statistics (W. Franks et al., Biomedical Engineering, IEEE Transactions on, vol. 52, no. 7, pp. 1295-1302, 2005).

(27) Moreover it should also be stressed that the finally recorded difference value is not equal to since the measured voltage value is also affected by the Ag/AgCl reference electrode potential.

(28) B. Media Only Voltage Measurements

(29) In this subsection the role of the area A and the capacitance C.sub.IA.sup.+ is investigated by means of the setup of FIG. 1 when the tissue sample is absent, i.e. when only media is used. The motivation for this stems from the need to confirm qualitatively the dependence of the recorded potential difference upon which, in turn according to our aforementioned strong assumption, depends upon the area A and the capacitance C.sub.IA.sup.+.

(30) FIG. 3 illustrates the recorded potential difference when the tip of the tungsten electrode (the remainder of the electrode is insulated) is immersed progressively by 25%, 50%, 75% and 100% of its length within RPMI-1640 medium (Life Technologies, Carlsbad, Calif.). Observe that the recorded potential difference increases with depth. As the electrode's depth increases the electrode's area interfacing with the media also increases. The measurements of FIG. 3 reveal a saturating trend for increasing area values. Confirm a similar trend for (3): bearing in mind that the quantity is bounded, the quantity reaches the saturation value /C.sub.dl when A takes large values.

(31) Despite the fact that the capacitances C.sub.IA.sup.+ and C.sub.IA.sup. are not part of the immediate electrode-specimen environment, they become part of the measurement process. Recording of the measured potential difference values for different C.sub.IA.sup.+ values (i.e. for different IAs) and for the same tip depth would be impractical. Instead it is straightforward to apply a capacitor in parallel with C.sub.IA.sup.+ and investigate its effect upon the measured potential difference for a given tip depth (i.e. for a given A value). Such a capacitor was applied haptically and the potential difference was recorded before and after the application of the haptic capacitor. The recorded potential difference was reduced from just over 200 mV to around 170 mV when the haptic capacitor was applied. Equation (3) reveals a similar behaviour for when the apparent C.sub.IA.sup.+ value increases while the other terms remain constant.

(32) The surface charge density in equations (1) and (3) should depend upon the microstructural characteristics of the specimen under test which is part of the electrode-(electrolyte plus tissue specimen) interface (see FIG. 1). In order to investigate the role of during measurement, the RPMI-1640 media was diluted progressively by means of deionised water. The recorded potential difference value decreased with decreasing media concentration (and thus decreasing ) values and for the same electrode tip depth. Confirm from (3) that decreasing values lead to decreasing surface potential values.

(33) Based on these results and taking into consideration that the microstructures of cancerous and non-cancerous omentum differ (M. Lobikin et al., Physical Biology, vol. 9, no. 6, p. 065002, 2012) (which might lead to different a values), it was theorised that the setup of FIG. 1 might prove useful in differentiating between the two types of tissue by recording a different potential difference value for each case. The next section investigates the potential of the setup of FIG. 1 in detecting such tissue voltage differences.

(34) Measured Results

(35) Ovarian cancer is one of the leading gynaecological cancers in the UK. Around 7000 women are diagnosed every year. Omental tissue was chosen as the testing specimen since omentum is an organ that stores lipids and regulates peritoneal fluid and is the main location where ovarian cancer metastasizes. An omentectomy is normally performed as a surgical treatment for ovarian cancer.

(36) All omentum specimens used were excised during cytoreductive surgery and measurements were carried out no later than half an hour. Appropriate tissue collection ethical approval and approval for experiments were set in place. The protocol for the collection of cancerous and non-cancerous tissue potential difference data was designed as follows: 1. Bring the RPMI-1640 tissue culture media to room temperature. 2. Place the Ag/AgCl reference electrode in a 1 ml pipette which is fixed by an iron stand. Fix the tungsten working electrode with the same iron stand. 3. Place an omental specimen of appropriate size in a separate 1 ml pipette tip. Fix the pipette tip containing tissue in an iron stand and place the lower part of tip into a beaker containing the RPMI-1640 tissue culture media. Place the tungsten electrode into the tissue. 4. Connect both electrodes to the customized IA board (a 10-channel especially built instrument containing AD8420 IAs), whose outputs are connected to the data acquisition system. 5. Record data for a minimum of 2 minutes until the value stabilises. 6. Repeat the recording for different specimens or different spots of the same specimen. 7. Dispose of the specimen in an appropriate way.

(37) Potential-difference data have been recorded in accordance with the above protocol from media only, non-cancerous omentum samples and cancerous ones. The results are shown in FIGS. 4, 5 and 6.

(38) For the experiments reported in FIGS. 4, 5 and 6, omentum samples were taken from 15 different patients. As can be seen from FIG. 4, the voltages were higher in non-cancerous tissue than in cancerous tissue. The results were significant at p<0.05 using both Mann-Whitney U-test and t-test paired statistical tests. FIG. 5 is a box-and-whisker plot depicting the quartiles of the voltage values shown in FIG. 4. FIG. 6 shows the difference in voltage values between the non-cancerous and cancerous omentum in individual patients. Each shape/letter in FIG. 6 represents an individual patient. The reported voltage values in FIG. 6 are the amplified (G) ones, where G (=10) is the gain of the amplifier. This Figure therefore refers to voltages in volts, whereas FIGS. 4 and 5 refer to voltages in millivolts.

(39) Conclusions

(40) It should be stressed that the difference in voltage level values between the cancerous and the non-cancerous case corresponds to difference of potential differences. Given that the Ag/AgCl electrode and the media type is common in all experiments, it can be concluded that the recorded voltage level differences reflect a difference in tissue properties.

Example 2Measuring the Voltage of Paired Cancerous and Non-Cancerous Omental Tissue Using Experimental Setup Without Medium

(41) Experimental Setup

(42) In these experiments, the experimental setup shown in FIG. 7 was used.

(43) Measured Results

(44) The results of the experiments are shown in FIG. 8. As can be seen from the Figure, the cancerous tissue had a lower voltage than the non-cancerous tissue.

Example 3Measuring the Voltage of Various Tissues

(45) Experimental Setup

(46) The voltage of medium alone, non-cancerous omentum and various cancerous tissues (omentum, right ovary, rectal sigmoid, spleen, para-aortic lymph node, pelvic side wall) was tested. In these experiments, the experimental setup shown in FIG. 1 was used.

(47) Measured Results

(48) The results of the experiments are shown in FIG. 9. As can be seen from the Figure, the various cancerous tissues had low voltages.

Example 4Measuring Voltage in Materials Characterized by Different Densities or Different Ion Contents

(49) Background

(50) The goal of this experiment was to examine if the method we have developed for taking biopotential measurements in human tissues using a tungsten working electrode and a double junction Ag/AgCl reference electrode can identify voltage differences in materials characterized by different densities or different ion contents. We manipulated material density by changing agar concentration in a gel. Moreover, we manipulated material ion content by changing media concentration in a gel. Based on this strategy, we produced two sets of gels. In the first set, the gels contained the same concentration of agar [3% (w/v)] but different media concentrations. The first gel contained 100% (v/v) media (30 mL media), the second 50% (v/v) media (15 mL deionized water and 15 mL media) and the third 10% (v/v) media (27 mL deionized water and 3 mL media). In the second set, the gels contained the same media concentration (10 mL) but different agar concentrations. The first gel contained 1% (w/v) agar (0.1 g), the second 2% (w/v) agar (0.2 g), the third 3% (w/v) agar (0.3 g) and the forth 5% (w/v) agar (0.5 g).

(51) Experimental Setup

(52) In these experiments, the experimental setup shown in FIG. 10 was used.

(53) Results

(54) The results for the two sets of gels are presented in FIGS. 11 and 12.

(55) Conclusions

(56) According to FIG. 11, if there are more ions in the material, the voltage difference between the working and the reference electrode increases. According to FIG. 12, if the material is harder (more compact), the voltage difference between the working and the reference electrode decreases. This finding is in accordance with the biopotential measurements described in Examples 1 to 3 in human tissues where cancerous tissues (which are harder structures) exhibit lower biopotential values compared to non-cancerous tissues (which are softer structures). Based on the results presented above, we conclude that this method can identify successfully voltage differences in materials which have different densities or different ion contents.