Sensing device with multiple sensing areas and sensing method by multiple sensing areas

Abstract

A sensing device with multiple sensing areas is provided. The sensing device with multiple sensing areas includes a substrate; a sample region arranged on the substrate, and configured for receiving an analyte; and a plurality of sensing areas arranged in the sample region on the substrate, wherein each of the sensing areas is configured to electrically connect to a Field Effect Transistor respectively.

Description

SENSING DEVICE WITH MULTIPLE SENSING AREAS AND SENSINGMETHOD BY MULTIPLE SENSING AREASBACKGROUND OF THE INVENTION 1. Field of the Invention

[0001] The present disclosure relates to a sensing device and method, and in particular to a sensing device and method with multiple sensing areas.2. Description of the Related Art

[0002] Sensing device for sensing and measurement of various chemical and biological reactions and identification are widely applied in the field of chemistry and biology.

[0003] One of such sensing devices is a Field-effect transistor (FET), which is a powerful biosensor with excellent sensitivity and specificity and has the advantages of low-cost, real-time, and label-free biosensing applications. By modifying the surface of FET with a probe to detect targets, such as nucleic acid and proteins, the sensing device can directly translate the signal of the target on the FET surface into an electronic signal.

[0004] One type of FET for biosensing is ion-sensitive field effect transistor, oftendenoted in the relevant literature as an ISFET. An ISFET is an impedance transformation device that operates in a manner similar to that of a MOSFET (Metal Oxide Semiconductor Field Effect Transistor), and is particularly configured to selectively measure ion activity in a solution.

[0005] Similar to the operation of MOSFET, the operation of ISFET is based on the charge concentration modulation (channel conductance) formed by a MOS capacitance formed by a polysilicon gate, a gate oxide and a region of the well (e.g., N-type well) between the source and the drain. For example, when a negative voltage is applied across the gate and source regions, a channel is created at the interface of the region and the gate oxide by depleting this area of electrons.

[0006] Another type of sensing device such an Extended-Gate Field-Effect Transistor, often denoted in the relevant literature as an EGFET, is based on the ISFET and implemented by connecting an extended gate to the gate of a MOSFET using a metal wire (or other material with conductive properties), to connect the sensing area to the gate of MOSFET (or also ISFET).

[0007] The main applications of EGFET are the detection of ionic species, pH, and specifically biological molecules such as antigens, cells, nucleic acids, and proteins, through the functionalization of sensing surfaces in sensing area contacted to theextended-gate of EGFET. By combining EGFET with other biomolecule / ion- sensitive recognition elements or materials, bio / ion sensing can be achieved.BRIEF SUMMARY OF THE INVENTION

[0008] However, since the aforementioned FETs and / or ISFETs and EGFETs are highly sensitive to the object to be sensed, it is important to enhance the sensing sensitivity and accuracy of the sensing device, and / or to increase the sensing types of the sensing device to improving its broad application (e.g. ability to sense different analytes in a sensing device).

[0009] The structure of the sensing device and the configuration of the sensing region are usually limited by the size of the biochip, the size of sensing equipment, the volume of the solution to be measured (e.g. low concentration bio-sample), the resolution of the sensing sensitivity, and the manufacturing process of the biochip, and so on, thus the sensing region of the sensing device has a certain limitation of space.

[0010] To solve the above problems, and in order to improve the sensitivity of the sensing device and increase the sensing ability to different types of targets under the conditions of limited sensing region and limited size of the sensing device, thepresent disclosure provides a sensing device for a biochip, which is able to improve the sensitivity of the sensing under the conditions of limited sensing region and be able to sense multiple sensing objects by providing plural sensing areas.

[0011] It is an objective of the present disclosure to provide a sensing device with multiple sensing areas. The sensing device with multiple sensing areas includes a substrate; a sample region arranged on the substrate, and configured for receiving an analyte; and a plurality of sensing areas arranged in the sample region on the substrate, wherein each of the sensing areas is configured to electrically connect to a Field Effect Transistor respectively.

[0012] In an embodiment, each of the sensing areas is configured to electrically connect to a plurality of Field Effect Transistors respectively.

[0013] In an embodiment, the plurality of sensing areas are symmetrically arranged about a geometric center of the plurality of sensing areas.

[0014] In an embodiment, the plurality of sensing areas have an interval with each other, wherein the interval is a non-sensing area.

[0015] In an embodiment, the plurality of sensing areas is arranged in the form of a nine square grid.

[0016] In an embodiment, the number of the plurality of sensing areas is eight, andthe sensing areas are arranged around a non-sensing area.

[0017] In an embodiment, the non-sensing area comprises an integrated electrode.

[0018] In an embodiment, the plurality of sensing areas is configured to sense the same analyte with same functionalization for each sensing areas.

[0019] In an embodiment, the plurality of sensing areas is configured to sense the different analytes with different functionalizations for each sensing areas.

[0020] In an embodiment, the plurality of sensing areas is configured to output the sensing signals collectively.

[0021] In an embodiment, each of the sensing areas is configured to output the sensing signals independently.

[0022] In an embodiment, the sample region is configured to be divided into multiple sub-regions, each of the sub-regions comprising at least one of the sensing areas.

[0023] In an embodiment, the sample region is configured to be divided into a first sub-region and a second sub-region, wherein each of the first sub-region and the second sub-region comprise three of the sensing areas.

[0024] In an embodiment, the sample region comprises at least one side wall to isolate each of the sub-regions.

[0025] It is another objective of the present disclosure to provide a sensing method by multiple sensing areas. The sensing method by multiple sensing areas includes steps of: providing a substrate; receiving an analyte by a sample region arranged on the substrate; and sensing the analyte by a plurality of sensing areas arranged in the sample region on the substrate, wherein each of the sensing areas is configured to electrically connect to a plurality of Field Effect Transistors respectively.

[0026] In an embodiment, the plurality of sensing areas is configured to sense the same analyte with same functionalization for each sensing areas.

[0027] In an embodiment, the plurality of sensing areas is configured to sense the different analytes with different functionalizations for each sensing areas.

[0028] In an embodiment, the plurality of sensing areas is configured to output the sensing signals collectively.

[0029] In an embodiment, each of the sensing areas is configured to output the sensing signals independently.

[0030] In an embodiment, the sample region is configured to be divided into multiple sub-regions, each of the sub-regions comprising at least one of the sensing areas.

[0031] In an embodiment, the sample region is configured to be divided into a firstsub-region and a second sub-region, wherein each of the first sub-region and the second sub-region comprise three of the sensing areas.

[0032] In an embodiment, the sample region comprises at least one side wall to isolate each of the sub-regions.

[0033] In an embodiment, the plurality of sensing areas are symmetrically arranged about a geometric center of the plurality of sensing areas

[0034] In an embodiment, the plurality of sensing areas have an interval with each other, wherein the interval is a non-sensing area.

[0035] In an embodiment, the plurality of sensing areas is arranged in the form of a nine square grid.

[0036] In an embodiment, the number of the plurality of sensing areas is eight, and the sensing areas are arranged around a non-sensing area.

[0037] In an embodiment, the non-sensing area comprises an integrated electrode. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] FIG. 1 illustrates a sensing device with multiple sensing areas according to an embodiment of the present disclosure.

[0039] FIG. 2 illustrates a sensing device with multiple sensing areas electricallyconnect to a plurality of Field Effect Transistors according to an embodiment of the present disclosure.

[0040] FIG. 3 illustrates a sensing device with multiple sensing areas arranged symmetrically according to an embodiment of the present disclosure.

[0041] FIG. 4 illustrates a sensing device with multiple sensing areas with interval according to an embodiment of the present disclosure.

[0042] FIG. 5 illustrates a sensing device with multiple sensing areas arranged in the form of a nine square grid according to an embodiment of the present disclosure.

[0043] FIG. 6 illustrates a sensing device with multiple sensing areas arranged in the form of a nine square grid included an integrated electrode according to an embodiment of the present disclosure.

[0044] FIG. 7 illustrates a sensing device with multiple sensing areas arranged in the form of a nine square grid included an integrated electrode and pads according to an embodiment of the present disclosure.

[0045] FIG. 8 illustrates a sensing device with multiple sensing areas for sensing same analyte with same functionalization according to an embodiment of the present disclosure.

[0046] FIG. 9 illustrates a sensing device with multiple sensing areas for sensingdifferent analytes with different functionalizations according to an embodiment of the present disclosure.

[0047] FIG. 10 illustrates a sensing device with multiple sensing areas divided into multiple sub-regions according to an embodiment of the present disclosure.

[0048] FIG. 11 illustrates a sensing device with multiple sensing areas divided into multiple sub-regions according to an embodiment of the present disclosure.

[0049] FIG. 12 illustrates a sensing device with multiple sensing areas with side wall according to an embodiment of the present disclosure.

[0050] FIG. 13A - FIG. 13H illustrate the diagrams showing the experiment results represented the uniformity of the plurality of the sensing areas by using the sensing device shown in FIG. 6 according to an embodiment of the present disclosure.

[0051] FIG. 14A illustrates a sensing device with multiple sensing areas with side wall according to an embodiment of the present disclosure.

[0052] FIG. 14B - FIG. 14S illustrate the diagrams showing the experiment results of sensing analyte by sensing areas labeled with Zones by using sensing device shown in FIG. 14A according to an embodiment of the present disclosure.DETAILED DESCRIPTION OF THE INVENTION

[0053] To facilitate understanding of the objects, characteristics and effects of the present disclosure, embodiments together with the attached drawings for the detailed description of the present disclosure are provided.

[0054] In order to improve the sensitivity of the sensing device and increase the sensing ability to different types of targets under the conditions of limited sensing region and limited size of the sensing device, the present disclosure provides a sensing device for a biochip, which is able to improve the sensitivity of the sensing under the conditions of limited sensing region and be able to sense multiple sensing objects by providing plural sensing areas.

[0055] FIG. l is a drawing illustrate a sensing device 10 with multiple sensing areas300 according to an embodiment of the present disclosure. As shown in FIG. 1, a sensing device 10 with multiple sensing areas includes a substrate 100, a sample region 200 and a plurality of sensing areas 300. The sample region 200 is arranged on the substrate 100 and configured for receiving an analyte, for example, biological sample such as solutions containing peptides, molecules, proteins, DNA / RNA, chemicals or ion, etc. The analyte, biological sample or solution will not extend beyond the sample region 200. The plurality of sensing areas 300 is arranged in the sample region 200 on the substrate 100, wherein each of the sensing areas 300 isconfigured to electrically connect to a Field Effect Transistor (FET) respectively.

[0056] In some embodiments, the Field Effect Transistor includes a gate, a source and a drain. Each of the sensing areas 300 is configured to electrically connect to the gate of the FET. In some embodiments, the FET is arranged in the substrate 100. In some embodiments, the FET is arranged outside the substrate 100 and electrically connected to the sensing areas by conductive material such as metal wire, electrode, etc. The FET is configured to change the conductive properties between the source and the drain based on sensing the electrical changes at the gate. The sensing can be achieved by measuring the minor change of electrical properties between source and the drain, for example, to measure the change signal of current through the source and the drain according to the ion concentration change sensing on the sensing areas 300 by the gate.

[0057] In some embodiments, the substrate 100 includes the conductive material such as metal plate, metal wire to provide the conductive path for sensing area 300 to electrically connect to FET or other sensing components on the same substrate 100 or outside the substrate 100 such as the sensing equipment via multiple electrode respectively.

[0058] With the structure shown in FIG. 1, by arranging the plurality of sensingareas 300 in the sample region 200 on the substrate 100, the biological sample or chemical sample can be sensed by multiple sensing areas 300 simultaneously and multiple sensing signals for the sample can be obtained simultaneously also. Comparing to known sensing device with a sensing area in a sample region, the sensing device 10 with multiple sensing areas 300 according to an embodiment of the present disclosure provides multiple sensing signals from multiple sensing areas 300 in a sample region 200, which means in a same limited space of the sample region in a sensing device (e.g. a biochip), the sensing device with multiple sensing areas according to the embodiment of the present disclosure is able to provide more sensing signals, and thus, is able to improve the sensitivity of the sensing device and increase the sensing ability to different types of targets by obtaining and / or analyzing the multiple sensing signals from multiple sensing areas in a sample region. For example, the sensing signal from one sensing area might be affected by noise, fluctuation or interference. In contrast, by using sensing device with multiple sensing areas according to the embodiment of the present disclosure, the multiple sensing signals can be obtained and analyzed, and the impacts from some noise, fluctuation or interference can be reduced.

[0059] FIG. 2 is a drawing illustrate a sensing device 10 with multiple sensing areas300 electrically connect to a plurality of Field Effect Transistors according to an embodiment of the present disclosure. In some embodiments, each of the sensing areas 300 is configured to electrically connect to a plurality of Field Effect Transistors respectively. As shown in FIG. 2, the sensing device 10 includes four sensing areas 300 in the sample region 200. Each sensing area 300 is further be divided into four sub-areas 310 and each sub-areas 310 is configured to electrically connect to one FET (e.g. gate of EGFET). That is, in addition to divide the sample region 200 into multiple independent sensing areas 300, each of the sensing areas 300 further divide into multiple sub-areas 310 and each sub-area 310 is electrically connect to the FET respectively, and the sensing signals from the sample region 200 further increased. Thus, by collecting and analyzing the signals from multiple subareas 310, the sensitivity of the sensing device can be further improved.

[0060] FIG. 3 is a drawing illustrate a sensing device 10 with multiple sensing areas 300 arranged symmetrically according to an embodiment of the present disclosure. In some embodiments, the plurality of sensing areas 300 are symmetrically arranged about a geometric center GC of the plurality of sensing areas 300. As shown in FIG. 3, the geometric center GC is in the center of the plurality of the sensing areas 300. The geometric center GC is defined by the layout of the plurality of the sensing areas300 but not sample region 200, however, sometimes the geometric center of the plurality of the sensing areas 300 and the geometric center of the sample region 200 can be same.

[0061] With the plurality of sensing areas 300 are symmetrically arranged about the geometric center GC of the plurality of sensing areas 300, due to the symmetrical layout, the sensing property of one sensing area will be the same as the other one symmetrical sensing area, thus the sensing properties, sensing quality and / or sensing reliability of the sensing areas 300 can be as consistent as possible. Accordingly, some signal fluctuation or interference during the operation of sensing, sensing preparation on the sensing device and / or some biological process could be reduced by analyzing the consistent sensing signals from symmetrical sensing areas.

[0062] FIG. 4 is a drawing illustrate a sensing device 10 with multiple sensing areas 300 with interval 350 according to an embodiment of the present disclosure. In some embodiments, the plurality of sensing areas 300 have an interval 350 with each other, wherein the interval 350 is a non-sensing area. As shown in FIG. 4, each sensing area 300 is not just adjacent to other sensing area 300, and the intervals 350 exist between every sensing area 300. The interval 350 is a non-sensing area, which means the interval 350 does not electrically connect to any FET, and the interval 350 is notformed by conductive material, however, the non-sensing feature of the interval 350 is for its surface. In some embodiments, some wires, FETs or electrical components electrically connected to the sensing areas 300 are provided in the substrate 100 under the interval 350. With the interval 350 separating the sensing areas 300, the sensing signals between each sensing areas 300 will not interfere with each other. In some embodiments, the material of the interval 350 is same as the material of the substrate 100. In some embodiments, the interval 350 is part of the substrate 100.

[0063] In some embodiments, the number of the plurality of sensing areas 300 is eight, and the sensing areas 300 are arranged around a non-sensing area, for example the space in the center of the plurality of sensing areas 300 shown in FIG. 4.

[0064] FIG. 5 is a drawing illustrate a sensing device 10 with multiple sensing areas 300 arranged in the form of a nine square grid according to an embodiment of the present disclosure. In some embodiments, the plurality of sensing areas 300 is arranged in the form of a nine square grid. Thereby, the sensing areas 300 can be arranged compactly and closely with each other, and the space utilization of the sample region 200 can be maximized while maintaining the advantages of symmetrical arrangement.

[0065] In some embodiments, each sensing area 300 can be numbered / labeled toknow which sensing area 300 the obtained sensing signal comes from. In some embodiments, some necessary electrical components for sensing can be located anywhere as shown in the aforementioned figures, such as around the sample region 200.

[0066] FIG. 6 is a drawing illustrate a sensing device 10 with multiple sensing areas300 arranged in the form of a nine square grid included an integrated electrode 400 according to an embodiment of the present disclosure. In some embodiments, the number of the plurality of sensing areas 300 is eight, and the sensing areas 300 are arranged around a non-sensing area. In some embodiments, the non-sensing area includes an integrated electrode 400, which contain some necessary components for sensing. Thereby, the sensing areas 300 can be arranged compactly and closely with each other, and the space utilization of the sample region 200 can be maximized while maintaining the advantages of symmetrical arrangement such as the consistency and / or reliability of sensing signals by arranging the sensing areas 300 about the integrated electrode 400.

[0067] FIG. 7 is a drawing illustrate a sensing device 10 with multiple sensing areas 300 arranged in the form of a nine square grid included an integrated electrode 400 and pads 500 according to an embodiment of the present disclosure. In someembodiments, the sensing device 10 includes several pads 500 arranged around and outside the sample region 200 for some electrical connection requirements.

[0068] FIG. 8 is a drawing illustrate a sensing device 10 with multiple sensing areas 300 for sensing same analyte with same functionalization according to an embodiment of the present disclosure. In some embodiments, the plurality of sensing areas 300 is configured to sense the same analyte with same functionalization (for example, denoted as ‘A’ in each sensing areas 300 in FIG. 8) for each sensing areas 300. In some embodiments, the plurality of sensing areas 300 is configured to output the sensing signals collectively. In some embodiments, one example for the same functionalization for each sensing areas 300 is treated the surface of the sensing areas 300 with same biological probe for sensing same analyte (same targeted sample). With the same functionalization for each sensing areas 300, multiple sensing signals which expected be consistent will be obtained, therefore, the accuracy of sensing can be improved. Also, with the sensing signals are output collectively, the sensing signals from multiple sensing areas 300 will be analyze simultaneously to reduce the impact form potential sensing error.

[0069] FIG. 9 is a drawing illustrate a sensing device 10 with multiple sensing areas 300 for sensing different analytes with different functionalizations according to anembodiment of the present disclosure. In some embodiments, the plurality of sensing areas is configured to sense the different analytes with different functionalization (for example, denoted as ‘A’, ‘B’ and ‘C’ in different sensing areas 300 in FIG. 9) for each sensing areas 300. In some embodiments, each of the sensing areas is configured to output the sensing signals independently. In some embodiments, one example for the different functionalizations for each sensing areas 300 are treated the surfaces of the sensing areas 300 with different biological probes for sensing different analytes (different targeted samples), such as some sensing areas 300 treated with antibodies for capturing proteins and some other sensing areas 300 treated with biotin for capturing single molecules. With the different functionalizations for each sensing areas 300, the sensing device 10 is able to sense different analytes in one sample solution receiving by the sample region 200 simultaneously. Thus, the sensing device 10 is able to sense different types of targets in one limited size sample region at same time depending on the sensing requirements.

[0070] In some embodiments, in addition to functionalize the sensing areas 300 for same or different sensing target, sensing areas 300 can be also functioned as “sensing reference” or “blank sensing” to provide different types of sensing signals for further data analysis and / or other quantitative analysis and processing.

[0071] FIG. 10 and FIG. 11 are drawings illustrate a sensing device 10 with multiple sensing areas 300 divided into multiple sub-regions according to an embodiment of the present disclosure. In some embodiments, the sample region 200 is configured to be divided into multiple sub-regions (for example, denoted as ‘710’ and ‘720’ in FIG. 10 or FIG. 11), each of the sub-regions including at least one of the sensing areas 300. With the ‘groups’ (i.e. sub-regions) of the sensing areas 300, the different sub-regions further provide the flexibility and can be applied for different functionalizations and / or different labels for border applications of the sensing device 10.

[0072] In some embodiments, the sample region is configured to be divided into a first sub-region 710 and a second sub-region 720, wherein each of the first sub-region 710 and the second sub-region 720 includes three of the sensing areas 300. Accordingly, with the arrangement of first sub-region 710 and a second sub-region 720, the sensing device 10 has the advantages of ‘groups’ applications while maintaining the benefits of making each sensing areas 300 uniform and symmetrical each other.

[0073] FIG. 12 is a drawing illustrate a sensing device 10 with multiple sensing areas 300 with side wall 800 according to an embodiment of the present disclosure.In some embodiments, the sample region 200 includes at least one side wall 800 to isolate each of the sub-regions 710, 720. In some embodiments, the side wall 800 is waterproof material and / or low-reactivity materials which will not affected the biological sample or sensing areas 300 such as material of substrate 100 or double- sided tape for experiment use, etc. In some embodiments, the side wall 800 is made during the manufacturing process of the sensing device 10. In some embodiments, the side wall 800 can be set according to the requirement during use of the sensing device 10. In some embodiments, as shown in FIG. 12, the side wall 800 is arranged in the middle and divides the sample region 200 into two sides, with each side has three sensing areas 300 symmetrically arranged. Therefore, the sensing device 10 can maintain its advantages of symmetrical and partitioned layout while ensuring that both sides (different sub-regions) are independent, isolated, and do not interfere with each other, so that the sensing device according to the embodiment of the present disclosure can be widely used, and can output signals collectively or independently to improve sensing accuracy, and can be applied to different types of biological targets and / or treated with different surface functionalizations.

[0074] FIG. 13A - FIG. 13H are the diagrams showing the experiment results represented the uniformity of the plurality of the sensing areas by using the sensingdevice with multiple sensing areas (M0-M7) arranged in the form of a nine square grid included an integrated electrode in the center shown in FIG. 6 according to an embodiment of the present disclosure.

[0075] In some embodiments, the experiment results are acquired by eight sensing areas (M0-M7) shown in FIG. 13A - FIG. 13H, each sensing areas electrically connects the gate of at least one FET respectively. The detection principle is similar to ISFET or EGFET by adjusting the gate potential through the electrochemical potential change between the electrolyte and the sensing areas. Therefore, it changes the channel conductance between the source and drain of the FET and affects the drain current. The ID-VG signal is affected by the charges collected by the floating gate and through affecting the conductance in the channel, and resulting in a shift in the electrical curve (Guliga et al., Extended gate field effect transistor (EGFET) integrated readout interfacing circuit for pH sensing, in 2014 2nd International Conference on Electrical, Electronics and System Engineering (ICEESE). 2014; Dorfman et al., Biomicrofluidics, 2020. 14(1): p. 011301). The sensor has a constant drain voltage (VD) of 2V and a gate voltage (VG) sweep of 0 V to 3 V is applied to the EGFET sensor. The electrical signal is measured as ID-VG curve and converted into the Vout-Vo curve through the formula (1). The difference between the area ofbaseline with each concentration is calculated.

[0076] Vout = IDxR (formula 1), in which R is the electrical resistance value.

[0077] In some embodiments, the samples are pH6 standard solution and pH7 standard solution. As shown in FIG. 13A - FIG. 13H, the results show the stability, consistency, and reliability of electrical measurement results of different sample by arranging multiple sensing areas (M0-M7) symmetrically, wherein the horizontal axis is VG and vertical axis is VD. Meanwhile, by using multiple sensing areas to collect multiple sets of signals at one time, which is able to compare and / or confirm the condition and / or any differences between each sensing area. For example, if there are defects in the manufacturing process of the device or uneven surface treatment, such as improper sample inflow position, surface tension, hydrophobicity, etc., such problems can be found and solved by using the sensing device with multiple sensing areas according to an embodiment of the present disclosure.

[0078] FIG. 14A is a drawing illustrate a sensing device 10 with multiple sensing areas 300 with side wall 800 according to an embodiment of the present disclosure.The sensing areas 300 are divided into isolated sub-regions 710 and 720 and labeled by Zone0-Zone2 in sub-regions 710 and Zone5-Zone7 in sub-regions 720.

[0079] FIG. 14B - FIG. 14S are the diagrams showing the experiment results ofsensing analyte (peptide) by sensing areas labeled with Zone0-Zone2 and Zone5-Zone7 by using sensing device 10 with multiple sensing areas 300 with side wall 800 shown in FIG. 14A according to an embodiment of the present disclosure.

[0080] In some embodiments, the Zone0-Zone2 and Zone5-Zone7 are isolated by side wall 800, the samples sensed at Zone0-Zone2 and Zone5-Zone7 will not affect each other. In some embodiments, the sensing areas (Zone0-Zone2 and Zone5-Zone7) are functionalized by APTES-biotin, and the symbols “B” in FIG. 14B - FIG. 14S show the sensing results after the step burn in the APTES-biotin to the surface of sensing areas (Zone0-Zone2 and Zone5-Zone7). Next, treat the neutravidin (N- Avidin) to the surface, the symbols “N” in FIG. 14B - FIG. 14S show the sensing results after the step treating the N-Avidin to the surface of sensing areas (ZoneO- Zone2 and Zone5-Zone7). Then, treat the biotinylated antibody (231-478 mAb) to the surface, the symbols “M” in FIG. 14B - FIG. 14S show the sensing results after the step treating the biotinylated antibody to the surface of sensing areas (ZoneO- Zone2 and Zone5-Zone7). After that, add the peptide (p-tau 231 peptide) as analyte to the sensing areas, the symbols “P” in FIG. 14B - FIG. 14S show the sensing results after the step adding the peptide to the surface of sensing areas (Zone0-Zone2 and Zone5-Zone7). Wherein, the “B-N”, “N-M” and “M-P” show the signal shiftcomparisons of sensing results between each step, as shown in FIG. 14B - FIG. 14S, the signal shifts between each zone are uniform and stable, which also show the stability, consistency and reliability of electrical measurement results of biological analytes with functionalization by using the sensing device with multiple sensing areas according to an embodiment of the present disclosure.

[0081] The present disclosure further provides a sensing method by multiple sensing areas. The sensing method by multiple sensing areas includes steps of: providing a substrate; receiving an analyte by a sample region arranged on the substrate; and sensing the analyte by a plurality of sensing areas arranged in the sample region on the substrate, wherein each of the sensing areas is configured to electrically connect to a plurality of Field Effect Transistors respectively.

[0082] In some embodiments, the plurality of sensing areas is configured to sense the same analyte with same functionalization for each sensing areas.

[0083] In some embodiments, the plurality of sensing areas is configured to sense the different analytes with different functionalizations for each sensing areas.

[0084] In some embodiments, the plurality of sensing areas is configured to output the sensing signals collectively.

[0085] In some embodiments, each of the sensing areas is configured to output thesensing signals independently.

[0086] In some embodiments, the sample region is configured to be divided into multiple sub-regions, each of the sub-regions comprising at least one of the sensing areas.

[0087] In some embodiments, the sample region is configured to be divided into a first sub-region and a second sub-region, wherein each of the first sub-region and the second sub-region comprise three of the sensing areas.

[0088] In some embodiments, the sample region comprises at least one side wall to isolate each of the sub-regions.

[0089] In some embodiments, the plurality of sensing areas are symmetrically arranged about a geometric center of the plurality of sensing areas

[0090] In some embodiments, the plurality of sensing areas have an interval with each other, wherein the interval is a non-sensing area.

[0091] In some embodiments, the plurality of sensing areas is arranged in the form of a nine square grid.

[0092] In some embodiments, the number of the plurality of sensing areas is eight, and the sensing areas are arranged around a non-sensing area.

[0093] In some embodiments, the non-sensing area comprises an integratedelectrode.

[0094] While the present disclosure has been described by means of specific embodiments, numerous modifications and variations could be made thereto by those skilled in the art without departing from the scope and spirit of the present disclosure set forth in the claims.

Claims

AMENDED CLAIMS received by the International Bureau on 10 January 2025 (10.01.2025)This is the replacement sheets containing a complete set of claims in replacement of the claims originally filed.

1. A sensing device with multiple sensing areas, comprising: a substrate; a sample region arranged on the substrate, and configured for receiving an analyte; and a plurality of sensing areas arranged in the sample region on the substrate, wherein each of the sensing areas is configured to electrically connect to a Field Effect Transistor respectively, and wherein each of the sensing areas is configured to electrically connect to different Field Effect Transistors respectively.

2. The sensing device with multiple sensing areas of claim 1, wherein each of the sensing areas is configured to electrically connect to a plurality of Field Effect Transistors respectively.

3. The sensing device with multiple sensing areas of claim 1, wherein the plurality of sensing areas are symmetrically arranged about a geometric center of the plurality of sensing areas.

4. The sensing device with multiple sensing areas of claim 1, wherein the plurality of sensing areas have an interval with each other, wherein the interval is a non-sensing area.

5. The sensing device with multiple sensing areas of claim 1, wherein the plurality of sensing areas is arranged in the form of a nine square grid.

6. The sensing device with multiple sensing areas of claim 5, wherein the number of the plurality of sensing areas is eight, and the sensing areas are arranged around a31AMENDED SHEET (ARTICLE 19)non-sensing area.

7. The sensing device with multiple sensing areas of claim 6, wherein the non-sensing area comprises an integrated electrode.

8. The sensing device with multiple sensing areas of claim 1, wherein the plurality of sensing areas is configured to sense the same analyte with same functionalization for each sensing areas.

9. The sensing device with multiple sensing areas of claim 1, wherein the plurality of sensing areas is configured to sense the different analytes with different functionalizations for each sensing areas.

10. The sensing device with multiple sensing areas of claim 1, wherein the plurality of sensing areas is configured to output the sensing signals collectively.

11. The sensing device with multiple sensing areas of claim 1, wherein each of the sensing areas is configured to output the sensing signals independently.

12. The sensing device with multiple sensing areas of claim 1, wherein the sample region is configured to be divided into multiple sub-regions, each of the sub-regions comprising at least one of the sensing areas.

13. The sensing device with multiple sensing areas of claim 12, wherein the sample region is configured to be divided into a first sub-region and a second sub-region, wherein each of the first sub-region and the second sub-region comprises three of the sensing areas.

14. The sensing device with multiple sensing areas of claim 12, wherein the sample region comprises at least one side wall to isolate each of the sub-regions.

15. A sensing method by multiple sensing areas, comprising steps of: providing a substrate; receiving an analyte by a sample region arranged on the substrate; and sensing the analyte by a plurality of sensing areas arranged in the sample32AMENDED SHEET (ARTICLE 19)region on the substrate, wherein each of the sensing areas is configured to electrically connect to a plurality of Field Effect Transistors respectively, and wherein each of the sensing areas is configured to electrically connect to different Field Effect Transistors respectively.

16. The sensing method by multiple sensing areas of claim 15, wherein the plurality of sensing areas is configured to sense the same analyte with same functionalization for each sensing areas.

17. The sensing method by multiple sensing areas of claim 15, wherein the plurality of sensing areas is configured to sense the different analytes with different functionalization for each sensing areas.

18. The sensing method by multiple sensing areas of claim 15, wherein the plurality of sensing areas is configured to output the sensing signals collectively.

19. The sensing method by multiple sensing areas of claim 15, wherein each of the sensing areas is configured to output the sensing signals independently.

20. The sensing method by multiple sensing areas of claim 15, wherein the sample region is configured to be divided into multiple sub-regions, each of the sub-regions comprising at least one of the sensing areas.33AMENDED SHEET (ARTICLE 19)Statement under Article 19(1)Regarding claim 1Claim 1 is amended according to the paragraphs [0074] - [0077] and Figs. 13A- 13H of the present application as filed.Claim 1 is amended to include “each of the sensing areas is configured to electrically connect to different Field Effect Transistors respectively”, based on the diagrams showing the experiment results of each of the sensing areas electrically connected to different Field Effect Transistors respectively (each sensing area corresponds to different MOSs) according to the paragraphs [0074] - [0077] and Figs. 13A-13H of the present application.WearOptimo is silent on each of the sensing areas electrically connected to different Field Effect Transistors respectively. WearOptimo (Figs. 3B-3C, paras [0401], [0470], [0478]) discloses “connectors 315 are provided which are connected to respective microstructures 312 via connections 313”, where microstructures are connected to same connector. However, WearOptimo fails to disclose each microstructure connected to different connectors respectively, that is, WearOptimo fails to disclose “each of the sensing areas is configured to electrically connect to different Field Effect Transistors respectively” recited in the amended claim 1.In addition, regarding claim 1, it is alleged in Written Opinion that WearOptimo discloses “a sample region arranged on the substrate, and configured for receiving an analyte” (Figs. 3B-3C, paras [0329], [0393], [0470]).Applicant respectfully clarifies that in WearOptimo, the microstructures is configured to interact, bind with analytes by breaching a functional barrier (claim 1 of WearOptimo), but not a chip with sample region for receiving analyte. WearOptimo is silent on providing sample region arranged on the substrate and configured for receiving an analyte.Regarding claim 15Claim 15 is amended according to the paragraphs [0074] - [0077] and Figs. 13A- 13H of the present application as filed.Claim 15 is amended to include “each of the sensing areas is configured to electrically connect to different Field Effect Transistors respectively”, based on the diagrams showing the experiment results of each of the sensing areas electrically connected to different Field Effect Transistors respectively.WearOptimo is silent on each of the sensing areas electrically connected to different Field Effect Transistors respectively. WearOptimo (Figs. 3B-3C, paras [0401], [0470], [0478]) discloses “connectors 315 are provided which are connected to respective microstructures 312 via connections 313”, where microstructures are connected to same connector. However, WearOptimo fails to disclose each microstructure connected to different connectors respectively, that is, WearOptimo fails to disclose “each of the sensing areas is configured to electrically connect to different Field Effect Transistors respectively” recited in the amended claim 15.In addition, regarding claim 15, it is alleged in Written Opinion that WearOptimo discloses “a sample region arranged on the substrate, and configured for receiving an analyte” (Figs. 3B-3C, paras [0329], [0393], [0470]).Applicant respectfully clarifies that in WearOptimo, the microstructures is configured to interact, bind with analytes by breaching a functional barrier (claim 1 of WearOptimo), but not a sample region for receiving analyte. WearOptimo is silent on providing sample region arranged on the substrate and configured for receiving an analyte.

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