DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Continued Examination
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on October 17, 2025 has been entered.
Status of the Claims
Claims 1, 5-7, 12, 22, 27-28, and 30 have been amended; claims 2, 8, 11, 14, 17, 23, 29, and 32 have been cancelled; and claims 15-16 and 18-21 have been withdrawn. Claims 1, 3-7, 9-10, 12-13, 15-16, 18-22, 24-28, 30-31 and 33-34 are currently pending and claims 1, 3-7, 9-10, 12-13, 22, 24-28, 30-31 and 33-34 are examined herein.
Status of the Rejection
Applicant’s amendments to the Claims have overcome each objection and 112(a) and 112(b) rejections previously set forth in the Final Office Action mailed July 17, 2025.
New grounds of claim objection are necessitated by the amendment.
New grounds of claim rejection under 35 U.S.C. § 112(b) are necessitated by the amendment.
All rejections under 35 U.S.C. § 103 from the previous office action are essentially maintained and modified in response to the amendment.
Claim Objection
Claims 1, 22 and 27 are objected to because of the following informalities:
Claims 1 and 22: please amend “the cell” to -- the at least one cell --.
Claim 27: please amend “an antibiotic drug; or an anticancer drug” to -- an antibiotic drug[[;]] or an anticancer drug --.
Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1, 3-7, 9-10, 12-13, 22, 24-28, 30-31 and 33-34 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as failing to set forth the subject matter which the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the applicant regards as the invention.
Regarding claim 1, claim 1 recites “removing the drug solution to form a cell free drug solution for obtaining a second electro-analytical measurement of the cell free drug solution”, which is unclear if a second electro-analytical measurement of the cell free drug solution is actually happing or not since it is not positively recited. Thus, the scope of claim 1 is indefinite. Claims 3-7, 9-10, and 12-13 are further rejected by virtue of their dependence upon and because they fail to cure the deficiencies of indefinite claim 1.
Regarding claim 22, claim 22 recites “removing the drug solution to form a cell free drug solution for obtaining a second electro-analytical measurement of the cell free drug solution”, which is unclear if a second electro-analytical measurement of the cell free drug solution is actually happing since it is not positively recited. Thus, the scope of claim 22 is indefinite. Claims 24-28, 30-31 and 33-34 are further rejected by virtue of their dependence upon and because they fail to cure the deficiencies of indefinite claim 22.
Claim Rejections - 35 USC § 103
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claims 1, 3-7, 9-10 and 12-13 are rejected under 35 U.S.C. 103 as being unpatentable over Gratzl et al. (WO2000073784A2), and in view of Yoshida et al. (US20050074869A1) and Abdolahad et al. (US20170176414A1).
Regarding claim 1, Gratzl teaches a method of determining drug permeability of a cell membrane (an electrochemical method for measuring drug efflux from a cell membrane [Ln 14 on page 9 to Ln 18 on page 10]), the method comprising:
(a) obtaining a biological sample (preparing the cells to be studied [Ln 19 on page 9]; the cell types include cancer cells, …, nerve cells [Ln 7-13 on page 9]);
(b) dispersing at least one cell from the biological sample to a discrete location or attached to a discrete substrate (one or more of the cells are deposited on a substrate and one or more different drugs studied, and the optimum dosage and/or type of drug for treating the patient’s condition is determined [Ln 22-25 on page 32]; cell is positioned on a substrate using hydrophilic regions [Ln 8-9 on page 32]; Fig.7 shows an array 80 of electrochemical cells 82 for simultaneous study of multiple cancer cells [Ln 25-29 on page 15]);
(c) exposing the at least one cell to one member of a drug panel in a drug solution, wherein the drug panel is composed of at least one drug at a concentration (charging [e.g., loading] cells with drugs [Ln 20 on page 9]; for doxorubicin, the drug concentration within 0.1-1.5 µM range is preferred [Ln 1-2 on page 18]; one or more of the cells are deposited on a substrate and one or more different drugs studied, and the optimum dosage and/or type of drug for treating the patient’s condition is determined [Ln 22-25 on page 32]);
(d) incubating the at least one cell from the biological sample in the drug solution for a given time (incubating cells with drugs [Ln 20 on page 9]; the cells are incubated in this drug-containing medium for a sufficient time for the cell or cells to absorb the drug. Typically, for doxorubicin, about 1h at 37 oC is sufficient [Ln 31-33 on page 13]); and
(f) removing the drug solution and adding a drug-less solution to the at least one cell (after loading/incubating cells with drugs, washing cells to remove drugs/other chemicals on the cell exterior, and immersing cells in test [efflux] medium [Ln 20-25 on page 9]; one suitable efflux medium comprises NaCl 140 mM, KCl 5.4 mM, Hepes 5.5 mM, CaCl2 2.5 mM, MgCl2 0.5 mM and glucose 11 mM adjusted to pH 7.4 witth 1 M NaOH [Ln 24-26 on page 10]; drug-free efflux medium [Ln 21-23 on page 34]. The drug-free efflux medium is deemed as the claimed drug-less solution).
Gratzl does not explicitly teach the following limitations: (1) the at least one drug at a plurality of concentrations; (2) obtaining a first electro-analytical measurement of the discrete location adjacent to the at least one cell; and (3) removing the drug solution to form a cell free drug solution for obtaining a second electro-analytical measurement of the cell free drug solution.
Gratzl further teaches one of the least well understood problems in cancer chemotherapy is the eventual resistance of tumor cells to different chemotherapeutic drugs of natural product origin, such as doxorubicin, actinomycin D, vinblastine, vincristine, or colchinine. Increasing the concentrations of one or more of these agents in small consecutive steps results in high- level cross-resistance in such cells to these as well as to many other, chemically unrelated drugs (the 2nd paragraph on page 1). One or more of the cells are deposited on a substrate and one or more different drugs studied, and the optimum dosage and/or type of drug for treating the patient’s condition is determined (Ln 22-25 on page 32).
Abdolahad teaches an electrochemical method for detecting effect of an anticancer drug on cancer cells (abstract), and further teaches the amount of change in electrical current due to the drug treatment may depend on the drug concentration, time of treatment, cells concentration, etc. [para. 0067].
Given the teachings of Gratzl regarding the concentrations of the one or more of these chemotherapeutic drugs affect the cross-resistance in such cells to these drugs; and one or more different drugs were studied to determine the optimum dosage and/or type of drug; and the teachings of Abdolahad regarding the drug concentration affecting drug treatment of cancer cells, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the at least one drug in the drug panel in Gratzl to provide a plurality of concentrations for the at least one drug in the drug panel, since it would allow to increase the concentrations of one or more of these agents (the at least one drug) in small consecutive steps resulting in high- level cross-resistance in such cells to these as well as to many other, chemically unrelated drugs (the 2nd paragraph on page 1 in Gratzl) and determine the optimum dosage of drug for treating the patient’s condition (the 5th paragraph on page 32 in Gratzl).
Modified Gratzl does not explicitly teach the following limitations: (2) obtaining a first electro-analytical measurement of the discrete location adjacent the at least one cell; and (3) removing the drug solution to form a cell free drug solution for obtaining a second electro-analytical measurement of the cell free drug solution.
Gratzl further teaches obtaining an electro-analytical measurement of the discrete location adjacent to the at least one cell for monitoring efflux from the cells using electrochemical monitoring equipment (steps 4a-4c in Ln27-31 on page 9). The detection methods are not limited to drug efflux, but can also be used for studying chemical influx. For example the movement of a chemical into a cell can be studies by immersing a cell in a culture medium containing the chemical and studying the increase in the chemical within the cell (Ln 3-7 on page 10). Such an arrangement can be used for studying efflux, influx, or influx and efflux simultaneously by using both internal and external electrodes/sensors (Ln 3-7 on page 24). The sensors may be spaced, for example to allow for study of variations in drug transport on the tissue level (Fig.14; Ln 32-35 on page 25).
Yoshida teaches a platform 33 employing electrochemical or optical means for studies of drug uptake and/or efflux of the cells. Drug uptake studies may include delivering a drug or other chemical of interest to the liquid medium or immersing the platform in a solution containing the drug, and studying uptake by the cells. Drug efflux (drug egress) studies may be carried out in a similar manner, but including further steps of removing the platform from the drug containing liquid after uptake has occurred and optionally washing the platform with a fresh, drug-free liquid to remove excess drug from the exterior of the cells. Efflux is then detected after placing the platform with the drug-containing cells in a fresh, drug-free liquid [para. 0062]. Thus, Yoshida teaches drug uptake and drug efflux of cells wherein the cells are, respectively, immersed in the drug solution and in a fresh drug-free liquid in the drug uptake and drug efflux studies.
Abdolahad teaches an electrochemical method for detecting effect of an anticancer drug on cancer cells, including: culturing a plurality of cancer cells to form cultured cells, attaching the cultured cells onto an array of silicon nanowires (SiNWs) electrodes, adding an anticancer drug to the attached cells onto the array of electrodes to form drug-treated cells, measuring a second electrochemical response of the drug-treated cells (abstract, Fig.3, [para. 0060]). Step 305- measuring a second electrochemical response of the drug-treated cells corresponds to the claimed step (e) of obtaining a first electro-analytical measurement of the at least one cell treated with the drug solution.
Given the teachings of Gratzl regarding the detection methods are not limited to drug efflux, but can also be used for studying chemical influx, and the electrochemical monitoring equipment can be used for studying efflux, influx, or influx and efflux simultaneously by using both internal and external electrodes/sensors; the teachings of Yoshida regarding drug uptake is detected by electrochemical or optical means after placing the cells in a drug solution [para. 0062, 0064 ]; and the teachings of Abdolahad regarding measuring an electrochemical response of the drug-treated cells to determine the effect of the drug on the cells (steps 304, 305, and 306 in Fig.3), it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the method in modified Gratzl by adding the step of obtaining a first electro-analytical measurement of the discrete location adjacent to the at least one cell, as taught by combined Gratzl, Yoshida, and Abdolahad, since it would allow for rapid screening of new drugs, and the uptake/efflux by cells/cell population (Ln 7-8 on page 32 in Gratzl); and would allow to study drug uptake by using the same platform for drug efflux [para. 0062 in Yoshida] and determine the effect of the anticancer drug on the cancer cells [para. 0060, 0066 in Abdolahad].
Modified Gratzl does not explicitly teach the following limitations: (3) removing the drug solution to form a cell free drug solution for obtaining a second electro-analytical measurement of the cell free drug solution.
Gratzl teaches remove drugs and other chemicals on the cell exterior prior to monitoring efflux form the cells using electrochemical monitoring equipment (Ln 22-28 on page 9).
Yoshida teaches dug efflux (drug egress) studies may be carried out in a similar manner, but including further steps of removing the platform from the drug containing liquid after uptake has occurred and optionally washing the platform with a fresh, drug-free liquid to remove excess drug from the exterior of the cells. Efflux is then detected after placing the platform with the drug-containing cells in a fresh, drug-free liquid [para. 0062].
Given the teachings of Gratzl regarding remove drugs and other chemicals on the cell exterior for efflux measurement; and the teachings of Yoshida regarding the cells are removed from the drug containing liquid, and then are placed in a fresh, drug-less liquid for detecting efflux, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the method in modified Gratzl by adding the step of removing the drug solution from the cells, as taught by combined Gratzl and Yoshida, since it would allow to perform the efflux measurement of the drug-containing cells [Ln 22-28 on page 9 of Gratzl; para. 0062 in Yoshida].
The limitation “to form a cell free drug solution for obtaining a second electro-analytical measurement of the cell free drug solution” is an intended result of a positively recited step. The court noted that a "‘whereby clause in a method claim is not given weight when it simply expresses the intended result of a process step positively recited.’" Id. (quoting Minton v. Nat’l Ass’n of Securities Dealers, Inc., 336 F.3d 1373, 1381, 67 USPQ2d 1614, 1620 (Fed. Cir. 2003)). MPEP 2111.04(I).
Regarding claim 3, modified Gratzl teaches the method of claim 1, and Gratzl teaches wherein the method further comprises incubating the at least one cell from the biological sample in the drug-less solution for a given time (preconcentration of effluxed drug at the electrode surface [step 4a in Ln 27-29 on page 9]).
Regarding claim 4, modified Gratzl teaches the method of claim 3, and Gratzl teaches wherein the method further comprises obtaining at least one further electro-analytical measurement of the discrete location adjacent to the at least one cell (Figs.1-2 show obtaining electro-analytical measurement of the discrete location adjacent the at least one cell 14; the working electrode 20, 50 is positioned in the efflux medium 28 at a selected distance from the cell 14 in the range of 0.5 – 2 µm [the 3rd paragraph on page 12]; measuring signals corresponding to drug concentration in step 4b of monitoring efflux from the cells using electrochemical monitoring equipment [Ln 27-30 on page 9]).
Regarding claim 5, modified Gratzl teaches the method of claim 1, and Gratzl teaches wherein the at least one drug is an electro-active drug (a selected electroactive drug 26, such as doxorubicin [Ln 17 on page 10]; Doxorubicin [also called Adriamycin], and some other anticancer drugs are "electroactive" [Ln 9-10 on page 4]).
Regarding claim 6, modified Gratzl teaches the method of claim 1, and Gratzl teaches wherein the at least one drug is an anticancer drug (Doxorubicin [also called Adriamycin], and some other anticancer drugs are "electroactive" [Ln 9-10 on page 4]).
Regarding claim 7, modified Gratzl teaches the method of claim 6, and Gratzl teaches wherein the at least one drug is one or more of the following: doxorubicin or daunorubicin (a selected electroactive drug 26, such as doxorubicin [Ln 17 on page 10]; the anticancer drug used in this latter was daunorubicin whose structure is very similar to doxorubicin [the 4th paragraph on page 26]).
Regarding claim 9, modified Gratzl teaches the method of claim 1, and Gratzl teaches wherein the drug panel is comprised of multiple drugs (one or more of the cells are deposited on a substrate and one or more different drugs studied, and the optimum dosage and/or type of drug for treating the patient’s condition is determined [Ln 22-25 on page 32]; for doxorubicin, the drug concentration within 0.1-1.5 µM range is preferred [Ln 1-2 on page 18]).
Gratzl is silent to wherein the multiple drugs each at a variety of concentrations.
Gratzl further teaches one of the least well understood problems in cancer chemotherapy is the eventual resistance of tumor cells to different chemotherapeutic drugs of natural product origin, such as doxorubicin, actinomycin D, vinblastine, vincristine, or colchinine. Increasing the concentrations of one or more of these agents in small consecutive steps results in high- level cross-resistance in such cells to these as well as to many other, chemically unrelated drugs (the 2nd paragraph on page 1). One or more of the cells are deposited on a substrate and one or more different drugs studied, and the optimum dosage and/or type of drug for treating the patient’s condition is determined (Ln 22-25 on page 32).
Abdolahad teaches the amount of change in electrical current due to the drug treatment may depend on the drug concentration, time of treatment, cells concentration, etc. [para. 0067].
Given the teachings of Gratzl regarding the concentrations of the one or more of these chemotherapeutic drugs affect the cross-resistance in such cells to these drugs; and one or more different drugs were studied to determine the optimum dosage and/or type of drug; and the teachings of Abdolahad regarding the drug concentration affecting drug treatment of the cancer cells, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the drug panel in modified Gratzl to provide a variety of concentrations for each drug of the multiple drugs in the drug panel, since it would allow to check the cross-resistance in the tumor cells to different chemotherapeutic drugs (the 2nd paragraph on page 1 in Gratzl) and determine the optimum dosage and/or type of drug for treating the patient’s condition (the 5th paragraph on page 32 in Gratzl).
Regarding claim 10, modified Gratzl teaches the method of claim 1, and Gratzl teaches wherein the biological sample comprises a cancer biopsy from a patient (a biopsy is performed on the patient to obtain cancer or other cells to be treated [the 5th paragraph on page 32]).
Regarding claim 12, modified Gratzl teaches the method of claim 1, wherein the first electro-analytical measurement is made by one or more of the following: cyclic voltammetry (CV) or differential pulse voltammetry (DPV) (the first and the second electrochemical responses may be for example, a cyclic voltammetry (CV) assay or a differential pulse voltammetry (DPV) response [para. 0068 in Abdolahad]; as outlined in the rejection of claim 1 above, the second electrochemical response of the drug-treated cells in Abdolahad is deemed as the claimed first electro-analytical measurement)
Regarding claim 13, modified Gratzl teaches the method of claim 5, and Gratzl teaches wherein electrode is optimized for the electro-active drug or electro-active drugs at the discrete location (a cylindrical carbon fiber microelectrode has been used to monitor doxorubicin efflux from AUXB1 and CHRC5 cell monolayers. The electrodes were found to be more stable and the signal was larger when doxorubicin reduction was used. Due to strong adsorption of the drug molecules onto the electrode surface, adsorption preconcentration could be employed to enhance the signal, i.e., the respective adsorption peak. By applying special electrochemical pretreatment, cleaning, and preconcentration protocols, doxorubicin concentrations down to 0.1 μM [in buffer with no live cells] could be detected [the 3rd paragraph on page 4]; between efflux measurements, the electrode is electrochemically preconditioned to regenerate the electrode surface and a preconcentration step is preferably carried out to preconcentrate the drug at the electrode surface to enhance the strength of the signal [Ln 10-13 on page 13]. Thus, Gratzl teaches wherein electrode is optimized for the electro-active drug).
Claims 22, 24-28, 30-31, and 33-34 are rejected under 35 U.S.C. 103 as being unpatentable over Gratzl et al. (WO2000073784A2), and in view of Yoshida et al. (US20050074869A1), Abdolahad et al. (US20170176414A1), and Lee et al. (Lab-free detection of single living bacteria via electrochemical collision event, Scientific Reports, 2016, 6, 30022).
Regarding claim 22, Gratzl teaches a method of determining membrane permeability of a cell to a drug (an electrochemical method for measuring drug efflux from a cell or cells [Ln 14 on page 9 to Ln 2 on page 10]), the method comprising:
(a) obtaining a biological sample (preparing the cells to be studied [Ln 19 on page 9]; the cell types include cancer cells, …, nerve cells [Ln 7-13 on page 9]);
(b) dispersing at least one cell from the biological sample to a surface or attached to a surface (one or more of the cells are deposited on a substrate and one or more different drugs studied, and the optimum dosage and/or type of drug for treating the patient’s condition is determined [Ln 22-25 on page 32]; cell is positioned on a substrate using hydrophilic regions [Ln 8-9 on page 32]; Fig.7 shows an array 80 of electrochemical cells 82 for simultaneous study of multiple cancer cells [Ln 25-27 on page 15]) ;
(c) exposing the at least one cell to a drug solution, wherein the drug solution has a given drug concentration (charging [e.g., loading] cells with drugs [Ln 20 on page 9]; for doxorubicin, the drug concentration within 0.1-1.5 µM range is preferred [Ln 1-2 on page 18]);
(d) incubating the at least one cell from the biological sample in the drug solution for a given time (incubating cells with drugs [Ln 20 on page 9]; the cells are incubated in this drug-containing medium for a sufficient time for the cell or cells to absorb the drug. Typically, for doxorubicin, about 1h at 37 oC is sufficient [Ln 31-33 on page 13]); and
(f) removing the drug solution and adding a drug-less solution to the at least one cell (after loading/incubating cells with drugs, washing cells to remove drugs/other chemicals on the cell exterior, and immersing cells in test [efflux] medium [Ln 20-25 on page 9]; one suitable efflux medium comprises NaCl 140 mM, KCl 5.4 mM, Hepes 5.5 mM, CaCl2 2.5 mM, MgCl2 0.5 mM and glucose 11 mM adjusted to pH 7.4 witth 1 M NaOH [Ln 24-26 on page 10]; drug-free efflux medium [Ln 21-23 on page 34]. The drug-free efflux medium is deemed as the claimed drug-less solution).
Gratzl is silent to the following limitations: (1) obtaining a first electro-analytical measurement of the at least one cell by impact chemistry (IC), whereby the at least one cell from the biological sample is made to collide with an electrode; (2) removing the drug solution to form a cell free drug solution for obtaining a second electro-analytical measurement of the cell free drug solution.
Gratzl further teaches obtaining an electro-analytical measurement of the at least one cell for monitoring efflux from the cells using electrochemical monitoring equipment (steps 4a-4c in Ln27-31 on page 9). The detection methods are not limited to drug efflux, but can also be used for studying chemical influx. For example the movement of a chemical into a cell can be studies by immersing a cell in a culture medium containing the chemical and studying the increase in the chemical within the cell (Ln 3-7 on page 10). Such an arrangement can be used for studying efflux, influx, or influx and efflux simultaneously by using both internal and external electrodes/sensors (Ln 3-7 on page 24). The sensors may be spaced, for example to allow for study of variations in drug transport on the tissue level (Fig.14; Ln 32-35 on page 25).
Yoshida teaches a platform 33 employing electrochemical or optical means for studies of drug uptake and/or efflux of the cells. Drug uptake studies may include delivering a drug or other chemical of interest to the liquid medium or immersing the platform in a solution containing the drug, and studying uptake by the cells. Drug efflux (drug egress) studies may be carried out in a similar manner, but including further steps of removing the platform from the drug containing liquid after uptake has occurred and optionally washing the platform with a fresh, drug-free liquid to remove excess drug from the exterior of the cells. Efflux is then detected after placing the platform with the drug-containing cells in a fresh, drug-free liquid [para. 0062]. Thus, Yoshida teaches drug uptake and drug efflux of cells wherein the cells are, respectively, immersed in the drug solution and in a fresh drug-free liquid in the drug uptake and drug efflux studies.
Abdolahad teaches an electrochemical method for detecting effect of an anticancer drug on cancer cells, including: culturing a plurality of cancer cells to form cultured cells, attaching the cultured cells onto an array of silicon nanowires (SiNWs) electrodes, adding an anticancer drug to the attached cells onto the array of electrodes to form drug-treated cells, measuring a second electrochemical response of the drug-treated cells (abstract, Fig.3, [para. 0060]). Step 305- measuring a second electrochemical response of the drug-treated cells corresponds to the claimed step (e) of obtaining a first electro-analytical measurement of the at least one cell treated with the drug solution.
Given the teachings of Gratzl regarding the detection methods are not limited to drug efflux, but can also be used for studying chemical influx, and the electrochemical monitoring equipment can be used for studying efflux, influx, or influx and efflux simultaneously by using both internal and external electrodes/sensors; the teachings of Yoshida regarding drug uptake is detected by electrochemical or optical means after placing the cells in a drug solution [para. 0062, 0064 ]; and the teachings of Abdolahad regarding measuring an electrochemical response of the drug-treated cells to determine the effect of the drug on the cells (steps 304, 305, and 306 in Fig.3), it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the method in Gratzl by adding the step of obtaining a first electro-analytical measurement of the at least one cell, as taught by combined Gratzl, Yoshida, and Abdolahad, since it would allow for rapid screening of new drugs, and the uptake/efflux by cells/cell population (Ln 7-8 on page 32 in Gratzl); and would allow to study drug uptake by using the same platform for drug efflux [para. 0062 in Yoshida] and determine the effect of the anticancer drug on the cancer cells [para. 0060, 0066 in Abdolahad].
Modified Gratzl is silent to: (1) wherein the first electro-analytical measurement is obtained by impact chemistry (IC), whereby the at least one cell from the biological sample is made to collide with an electrode; and (2) removing the drug solution to form a cell free drug solution for obtaining a second electro-analytical measurement of the cell free drug solution.
Lee teaches detecting single living bacterial cells on ultramicroelectrode (UME) using a single-particle collision method. The number of collision events involving the bacterial cells indicated in current-time (i-t) curves corresponds to the number of bacterial cells (i.e., Escherichia coli) on the UME surface. This single-particle collision approach facilitates detecting living bacteria and determining their concentration in solution and could be widely applied to studying other bacteria and biomolecules (abstract, Fig.1). The negatively charged E. coli is attracted to the UME surface through electrophoretic migration. When an E. coli collides with and then attaches to the UME surface, the level of the steady-state current decreases immediately because the flux of the redox species is blocked by the E. coli. Therefore, a staircase current response is observed (Fig. 1, the 1st paragraph on page 2).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the electrochemical method of measuring the first electro-analytical measurement of the at least one cell treated by the drug solution in modified Gratzl to obtain the first electro-analytical measurement of the at least one cell by impact chemistry (IC), whereby the at least one cell from the biological sample is made to collide with an electrode, as taught by Lee, since this single-particle collision approach would facilitate detecting the properties of single cell after separation and/or attachment onto the UME surface (abstract and conclusions in Lee).
Modified Gratzl does not explicitly teach the following limitations: (3) removing the drug solution to form a cell free drug solution for obtaining a second electro-analytical measurement of the cell free drug solution.
Gratzl teaches remove drugs and other chemicals on the cell exterior prior to monitoring efflux form the cells using electrochemical monitoring equipment (Ln 22-28 on page 9).
Yoshida teaches dug efflux (drug egress) studies may be carried out in a similar manner, but including further steps of removing the platform from the drug containing liquid after uptake has occurred and optionally washing the platform with a fresh, drug-free liquid to remove excess drug from the exterior of the cells. Efflux is then detected after placing the platform with the drug-containing cells in a fresh, drug-free liquid [para. 0062].
Given the teachings of Gratzl regarding remove drugs and other chemicals on the cell exterior for efflux measurement; and the teachings of Yoshida regarding the cells are removed from the drug containing liquid, and then are placed in a fresh, drug-less liquid for detecting efflux, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the method in modified Gratzl by adding the step of removing the drug solution from the cells, as taught by combined Gratzl and Yoshida, since it would allow to perform the efflux measurement of the drug-containing cells [Ln 22-28 on page 9 of Gratzl; para. 0062 in Yoshida].
The limitation “to form a cell free drug solution for obtaining a second electro-analytical measurement of the cell free drug solution” is an intended result of a positively recited step. The court noted that a "‘whereby clause in a method claim is not given weight when it simply expresses the intended result of a process step positively recited.’" Id. (quoting Minton v. Nat’l Ass’n of Securities Dealers, Inc., 336 F.3d 1373, 1381, 67 USPQ2d 1614, 1620 (Fed. Cir. 2003)). MPEP 2111.04(I).
Regarding claim 24, modified Gratzl teaches the method of claim 22, and Gratzl teaches wherein the method further comprises incubating the at least one cell from the biological sample in the drug-less solution for a given time (preconcentration of effluxed drug at the electrode surface [step 4a in Ln 27-29 on page 9]).
Regarding claim 25, modified Gratzl teaches the method of claim 24, and Gratzl teaches wherein the method further comprises obtaining at least one further electro-analytical measurement of the at least one cell from the biological sample (Figs.1-2 show obtaining electro-analytical measurement of the at least one cell 14; the working electrode 20, 50 is positioned in the efflux medium 28 at a selected distance from the cell 14 in the range of 0.5 – 2 µm [the 3rd paragraph on page 12]; measuring signals corresponding to drug concentration in step 4b of monitoring efflux from the cells using electrochemical monitoring equipment [Ln 27-30 on page 9]).
Regarding claim 26, modified Gratzl teaches the method of claim 22, and Gratzl teaches wherein the drug is an electro-active drug (a selected electroactive drug 26, such as doxorubicin [Ln 17 on page 10]; Doxorubicin [also called Adriamycin], and some other anticancer drugs are "electroactive" [Ln 9-10 on page 4]).
Regarding claim 27, modified Gratzl teaches the method of claim 22, and Gratzl teaches wherein the drug is an anticancer drug (Doxorubicin [also called Adriamycin], and some other anticancer drugs are "electroactive" [Ln 9-10 on page 4]).
Regarding claim 28, modified Gratzl teaches the method of claim 27, and Gratzl teaches wherein the drug is one or more of the following: doxorubicin or daunorubicin (a selected electroactive drug 26, such as doxorubicin [Ln 17 on page 10]; the anticancer drug used in this latter was daunorubicin whose structure is very similar to doxorubicin [the 4th paragraph on page 26]).
Regarding claim 30, modified Gratzl teaches the method of claim 22, and Gratzl teaches wherein the dispersing of the at least one cell from the biological sample to the surface or attached to the surface a drug panel is repeated on multiple discrete surfaces (multiple cancer cells 14 or groups of cancer cells using an array 80 of electrochemical cells 82 in Fig.7. A single cell 14, or a population of cells, is positioned in each of the wells on a hydrophilic area 96 [Ln 25 on page 15 to Ln 2 on page 16]; charging [e.g., loading] cells with drugs [Ln 20 on page 9]; for doxorubicin, the drug concentration within 0.1-1.5 µM range is preferred [Ln 1-2 on page 18]; one or more of the cells are deposited on a substrate and one or more different drugs studied, and the optimum dosage and/or type of drug for treating the patient’s condition is determined [Ln 22-25 on page 32]. Thus a drug panel is repeated on multiple discrete surfaces of the wells of the electrochemical cells 82) so that multiple drugs are available (provision of in-situ monitoring of drugs [Ln 23-24 on page 6]; one or more of the cells are deposited on a substrate and one or more different drugs studied, and the optimum dosage and/or type of drug for treating the patient’s condition is determined [Ln 22-25 on page 32]) .
Modified Gratzl further teaches a variety of concentrations for the drug of doxorubicin (doxorubicin of fixed concentrations of 0.2, 0.5 and 1 μM [the 3rd paragraph on page 35 in Gratzl]) available for IC electro-analytical measurement (Lee teaches the IC electro-analytical measurement, as outlined in the rejection of claim 22 above).
Gratzl is silent to wherein the multiple drugs at a variety of concentrations or combinations of drugs each at a variety of concentrations.
Gratzl further teaches one of the least well understood problems in cancer chemotherapy is the eventual resistance of tumor cells to different chemotherapeutic drugs of natural product origin, such as doxorubicin, actinomycin D, vinblastine, vincristine, or colchinine. Increasing the concentrations of one or more of these agents in small consecutive steps results in high- level cross-resistance in such cells to these as well as to many other, chemically unrelated drugs (the 2nd paragraph on page 1). One or more of the cells are deposited on a substrate and one or more different drugs studied, and the optimum dosage and/or type of drug for treating the patient’s condition is determined [Ln 22-25 on page 32].
Abdolahad teaches an electrochemical method for detecting effect of an anticancer drug on cancer cells (abstract), and further teaches the amount of change in electrical current due to the drug treatment may depend on the drug concentration, time of treatment, cells concentration, etc. [para. 0067].
Given the teachings of Gratzl regarding the concentrations of the one or more of these chemotherapeutic drugs affect the cross-resistance in such cells to these drugs; and one or more different drugs were studied to determine the optimum dosage and/or type of drug; and the teachings of Abdolahad regarding the drug concentration affecting drug treatment of cancer cells, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the drug panel in modified Gratzl to provide a variety of concentrations for each drug of the multiple drugs in the drug panel, since it would allow to check the cross-resistance in the tumor cells to different chemotherapeutic drugs (the 2nd paragraph on page 1 in Gratzl) and determine the optimum dosage and/or type of drug for treating the patient’s condition (the 5th paragraph on page 32 in Gratzl).
Regarding claim 31, modified Gratzl teaches the method of claim 22, and Gratzl teaches wherein the biological sample comprises a cancer biopsy from a patient (a biopsy is performed on the patient to obtain cancer or other cells to be treated [the 5th paragraph on page 32]).
Regarding claim 33, modified Gratzl teaches the method of claim 22, wherein the electrode is a wire electrode (the IC electrode used in Lee is a carbon fiber ultramicroelectrode [section of preparation of C-UMEs on page 5; Fig.1 in Lee], which is a wire electrode. Examiner notes the working electrode 20 in Fig.1 of Gratzl is also a carbon fiber microdiskelectrode [Ln 15-16 on page 10 in Gratzl], which is a wire electrode).
Regarding claim 34, modified Gratzl teaches the method of claim 26, wherein the electrode is optimized for the electro-active drug (the IC electrode used in Lee is a carbon fiber ultramicroelectrode [section of preparation of C-UMEs on page 5; Fig.1 in Lee]; the working electrode 20 in Fig.1 of Gratzl is also a carbon fiber microdiskelectrode [Ln 15-16 on page 10 in Gratzl]. Gratzl further teaches a cylindrical carbon fiber microelectrode has been used to monitor doxorubicin efflux from AUXB1 and CHRC5 cell monolayers. The electrodes were found to be more stable and the signal was larger when doxorubicin reduction was used. Due to strong adsorption of the drug molecules onto the electrode surface, adsorption preconcentration could be employed to enhance the signal, i.e., the respective adsorption peak. By applying special electrochemical pretreatment, cleaning, and preconcentration protocols, doxorubicin concentrations down to 0.1 μM [in buffer with no live cells] could be detected [the 3rd paragraph on page 4]; between efflux measurements, the electrode is electrochemically preconditioned to regenerate the electrode surface and a preconcentration step is preferably carried out to preconcentrate the drug at the electrode surface to enhance the strength of the signal [Ln 10-13 on page 13]. Thus, Gratzl teaches wherein the electrode, which is essentially the same as the IC electrode of Lee, is optimized for the electro-active drug of doxorubicin).
Response to Arguments
Applicant's arguments, see Remarks Pgs. 13-30, filed 10/17/2025, with respect to the 35 U.S.C. § 103 rejections have been fully considered, and the 103 rejections from the previous office action are modified in response to the amendment to claims.
Applicant’s Argument #1:
Regarding claims 1 and 22, Applicant argues at pages 27-29 that claims 1 and 22 are amended to specify "a first electro-analytical measurement" and "a second electro-analytical measurement" and to clarify that the "second electro-analytical measurement" is of "the cell free drug solution." As mentioned above in reference to Figures 10 and 11, and TABLE 3 of the present application the "cell free drug solution" is the "supernatant" that was collected. Whereby, any ciprofloxacin taken up by the bacteria during the incubation time was hence removed from the solution and the measurements performed on the "supernatant" would be an indication of ciprofloxacin uptake by the bacteria. Although Yoshida purports to test influx or drug uptake into the cells, the measurements are always on cells and never done on a cell-free drug solution that has been removed following incubation with a cell, as required by the present claims. None of Gratzl, Abdolohad, and/or Lee remedy this deficiency and thus cannot render the present claims obvious.
Examiner’s Response #1:
Applicant’s arguments have been fully considered, but are moot in view of the updated rejections for the amended claims 1 and 22 above. Examiner notes that “to form a cell free drug solution for obtaining a second electro-analytical measurement of the cell free drug solution” is an intended result of a positively recited step, and a second electro-analytical measurement of the cell free drug solution may not actually happen. Examiner suggests applicant to positively recite the step of a second electro-analytical measurement of the cell free drug solution.
Conclusion
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/SHIZHI QIAN/Examiner, Art Unit 1795