TWI664417B - Optical test strip and test method thereof for cancer cell/bacteria - Google Patents

Abstract

An optical cancer cell / bacterial detection test strip includes a high reflectance / transparent substrate, an ordered array structure layer, and a plurality of fixed substances. The ordered array structure layer is disposed on the high reflectivity / transparent substrate, and the ordered array structure layer includes a plurality of raised portions and a plurality of gaps. The gap is between the raised portions. The width of the raised portion is between 100 nm and 10,000 nm. The ratio of the height to the width of the raised portion is between 0.5 and 5. The ratio of the gap pitch to the width of the raised portion is between 0.2 and 8. The fixed substance is arranged on the raised portions of the ordered array structure layer to capture cancer cells / bacteria. When the laser light is irradiated to the test strip, the laser light is diffracted, and the laser diffraction intensity value is obtained as an index for detecting the cancer cells / bacteria on the surface of the optical cancer cell / bacterial detection strip.

Description

Optical cancer cell / bacterial detection test strip and detection method thereof

The invention relates to a detection test strip and a detection method thereof, and in particular to an optical cancer cell / bacterial detection test strip and a detection method thereof.

Generally, cancer patients often die from metastasis of cancer cells, and the method of metastasis is mainly performed by Circulating Tumor Cells in the circulatory system. Therefore, clinically, it is possible to reach the early stage by detecting circulating tumor cells in the blood. Discovery and the purpose of early treatment. However, because the number of circulating tumor cells in the early stages of cancer is relatively small, and the number of white blood cells will increase in the early stages of cancer, it often takes a long time to find circulating tumor cells in tens of thousands of white blood cells.

At present, although antibodies can be used to identify circulating tumor cells in the blood, and then magnetic beads, microfluidics, or a combination of these methods can be used to capture circulating tumor cells identified by the antibodies, fluorescence staining is still required confirm. In other words, with the current method, it still takes several hours to test a sample to get the result.

The invention provides an optical cancer cell / bacterial detection test strip, which has the advantages of simple manufacturing process and low manufacturing cost.

The invention also provides a method for detecting an optical cancer cell / bacterial detection test strip, which uses the above-mentioned detection test strip and has the effects of less sample demand and rapid detection.

An optical cancer cell / bacterial detection test strip of the present invention includes a high reflectance / transparent substrate, an ordered array structure layer, and a plurality of fixed substances. The ordered array structure layer is disposed on the high reflectivity / transparent substrate, and the ordered array structure layer includes a plurality of raised portions and a plurality of gaps. The gap is between the raised portions. The width of the raised portion is between 100 nm and 10,000 nm. The ratio of the height to the width of the raised portion is between 0.5 and 5. The ratio of the gap pitch to the width of the raised portion is between 0.2 and 8. The fixed substance is arranged on the raised portions of the ordered array structure layer to capture cancer cells / bacteria. When the laser light irradiates the optical cancer cell / bacteria test strip, the laser light will be diffracted and the laser diffraction intensity value will be obtained as an indicator for detecting the cancer cells / bacteria on the surface of the optical cancer cell / bacteria test strip. .

In an embodiment of the present invention, the above-mentioned optical cancer cell / bacterial detection test strip is a penetrating detection test strip, and the manner in which the laser light is diffracted includes passing the laser light through the optical cancer cell / bacterial detection test strip. Ordered array structure layer.

According to an embodiment of the present invention, a material of the transparent substrate includes polydimethylsiloxane (PDMS).

In an embodiment of the present invention, the protrusions and the gaps are arranged in a one-dimensional periodic pattern or a two-dimensional array pattern.

In an embodiment of the present invention, the one-dimensional periodic pattern includes a line shape and a groove.

In an embodiment of the invention, the two-dimensional array pattern includes a pillar shape and a hole.

In an embodiment of the present invention, the optical cancer cell / bacteria detection test strip is a reflection detection specimen, and the method of diffracting laser light includes ordering the laser light on the optical cancer cell / bacteria detection test strip in an orderly manner. Reflection on the array structure layer.

In an embodiment of the present invention, a material of the high-reflectivity substrate includes a silicon wafer.

In one embodiment of the present invention, the above-mentioned fixing substance includes an antibody.

In one embodiment of the present invention, the cancer cells / bacteria include circulating tumor cells or Yersinia pestis.

The method for detecting an optical cancer cell / bacterial detection test strip of the present invention includes the following steps. Provide the above-mentioned optical cancer cell / bacterial detection test strip and sample. The sample is dropped on the optical cancer cell / bacteria test strip, and the sample is reacted with the fixed substance of the optical cancer cell / bacteria test strip to obtain a reacted optical cancer cell / bacteria test strip. Provide laser light, so that the laser light irradiates the optical cancer cell / bacteria test strip after the reaction and detects the laser diffraction intensity value after the side laser light is diffracted. Compare the laser diffraction intensity value measured by the optical cancer cell / bacteria test strip after the reaction with the laser diffraction intensity value measured by the optical cancer cell / bacteria test strip before the sample is added. When the value of the laser diffraction intensity measured by the optical cancer cell / bacterial test strip after the reaction decreases, it means that the cancer cells / bacteria are contained in the blood.

In an embodiment of the present invention, the above-mentioned manner of diffracting the laser light includes passing the laser light through the ordered array structure layer of the optical cancer cell / bacterial detection test strip, or applying the laser light to the optical cancer cell / bacterial detection test. Sheets of the ordered array structure are reflected on layers.

In one embodiment of the present invention, the method for obtaining a sample includes centrifuging blood and removing a buffy coat.

In an embodiment of the present invention, before the laser light is provided, the method further includes adding a washing solution to remove cells in the sample that have not reacted with the fixed substance.

Based on the above, in the optical cancer cell / bacteria detection test strip and the detection method of the present invention, the optical cancer cell / bacteria detection test strip includes an ordered array structure layer and a fixed substance capable of grasping the cancer cells / bacteria. The width of the protrusions is between 100 nm and 10,000 nm. The ratio of the height of the protrusions to the width is between 0.5 and 5. The ratio of the gap distance to the width of the protrusions is between Between 0.2 and 8. Therefore, the laser light can be used to detect the optical cancer cell / bacterial detection test strip after reacting with the sample. When the laser diffraction intensity value measured by the reacted optical cancer cell / bacterial detection test strip decreases, then This indicates that the sample contains cancer cells / bacteria. With this design, the optical cancer cell / bacterial detection test strip provided by the present invention has the advantages of simple manufacturing process and low price. In addition, the method for detecting an optical cancer cell / bacteria test strip provided by the present invention can be used to detect cancer cells / bacteria in blood, and has the advantages of less sample demand and rapid detection.

In order to make the above features and advantages of the present invention more comprehensible, embodiments are hereinafter described in detail with reference to the accompanying drawings.

FIG. 1A is a schematic partial perspective view of an optical cancer cell / bacterial detection test strip according to an embodiment of the present invention. Fig. 1B is a schematic cross-sectional view of the optical cancer cell / bacteria detection test strip taken along line A-A 'of Fig. 1A. For clarity, some components are not shown in FIG. 1A. Please refer to FIGS. 1A and 1B. In this embodiment, the optical cancer cell / bacterial detection test strip 100 includes a transparent substrate 110, an ordered array structure layer 120, and a plurality of fixing substances 130. The material of the transparent substrate 110 may be, for example, a transparent polymer material and has a function of resisting biological adhesion.

In detail, in this embodiment, the ordered array structure layer 120 is disposed on the transparent substrate 110, the fixed substance 130 is disposed on the ordered array structure layer 120, and the fixed substance 130 and the transparent substrate 110 are respectively located on the ordered array structure. Opposite sides of the layer 120. The ordered array structure layer 120 includes a plurality of raised portions 122 and a plurality of gaps 124, and the gaps 124 are located between the raised portions 122. In this embodiment, the height of the raised portion 122 is H1, the height at H1 of 1/2 is H2, and the width of the raised portion 122 corresponding to the height H2 is defined as the width W1 of the raised portion 122, and The width of the gap 124 corresponding to the height H2 is defined as the pitch G1 of the gap 124. The width W1 of the raised portion 122 is between 100 nm and 10,000 nm. The ratio of the height H1 to the width W1 of the raised portion 122 is between 0.5 and 5. For example, in this embodiment, when the width W1 of the raised portion 122 is 500 nm, the height H1 of the raised portion 122 may be between 250 nm and 2500 nm. In some embodiments, when the width W1 of the raised portion is 1000 nm, the height H1 of the raised portion may be between 500 nm and 5000 nm.

Please continue to refer to FIGS. 1A and 1B. In this embodiment, the optical cancer cell / bacterial detection test strip 100 is a penetrating detection test strip. Therefore, when the laser light irradiates the optical cancer cell / bacterial detection test strip 100, the laser light will penetrate the ordered array structure layer 120 of the optical cancer cell / bacterial detection test strip 100, so that the laser light is diffracted and can be measured. The laser diffraction intensity value is used as an index for detecting cancer cells / bacteria on the surface of the optical cancer cell / bacterial detection test strip 100. Here, the material of the transparent substrate 110 may be, for example, polydimethylsiloxane (PDMS), but is not limited thereto. In some embodiments, the material of the transparent substrate 110 may also be polymethyl methacrylate (PMMA), polystyrene (PS), polycarbonate (PC), polyvinyl chloride, or polyethylene terephthalate. (PET) or other transparent polymer materials with anti-bioadhesion function.

In this embodiment, the protrusions 122 and the gaps 124 may be arranged in a one-dimensional periodic pattern. In detail, the convex portion 122 in FIG. 1A is embodied as a linear protrusion, and the gap 124 is embodied as a groove. The linear convex protrusion 122 and the groove-shaped gap 124 are along the first One direction X extends into a stripe pattern, and the linearly protruding protrusions 122 and the groove-shaped gaps 124 are arranged at intervals from each other in the second direction Y, and further arranged in a one-dimensional periodic pattern on the transparent substrate 110. It should be noted that, although in the ordered array structure layer 120 of the optical cancer cell / bacteria detection test strip 100 in this embodiment, the convex portions 122 and the gaps 124 may be arranged in a one-dimensional periodic pattern, the present invention is not limited to This is limited.

Please refer to FIG. 1B again. In this embodiment, the ratio of the pitch G1 of the gap 124 of the ordered array structure layer 120 to the width W1 of the convex portion 122 is, for example, 0.2 to 8, preferably, for example, Between 0.5 and 3. In this embodiment, the ratio obtained by dividing the pitch G1 of the gap 124 by the width W1 of the raised portion 122 is defined as the duty ratio of the ordered array structure layer 100. Therefore, the working ratio may be, for example, between 0.2 and 8, preferably, the working ratio may be, for example, between 0.5 and 3.

Therefore, in the present embodiment, when the width W1 of the raised portion 122 is 500 nm and the working ratio is 0.5, the pitch G1 of the corresponding gap 124 is 250 nm. That is, approximately 毎 750 nanometers (width W1 + pitch G1) will have one raised portion, 毎 1 cm will have 1.3´10 ⁴ raised portions 122, and an ordered array of 毎 1 square centimeter (cm ² ) In the structure layer 120, there are 1.3´10 ^four raised portions 122. Similarly, when the width W1 of the raised portion 122 is 500 nm and the working ratio is 8, after conversion, there will be 2.2´10 ³ raised portions 122 in the ordered array structure layer 120 of 1 cm 2. . In other words, in the ordered array structure layer 120 where the width W1 of the raised portion 122 is 500 nanometers, since the working ratio is between 0.5 and 5, the density range of the raised portion 122 is between 2.2´10 and ³ lines. / cm ² to 1.3´10 ⁴ bars / cm ² . It should be noted that, although the width W1 of the raised portion 122 is 500 nanometers as an example, the present invention is not limited thereto. In other embodiments, the width W1 of the raised portion may be less than or greater than 500. Nanometer, the calculated density range will also be different.

It is worth noting that when the working ratio is larger, the gap 124 between the two raised portions 122 is larger, and the distribution of the raised portions 122 in the ordered array structure layer 100 is more sparse. On the contrary, the smaller the working ratio is, the smaller the gap 124 between the raised portions 122 is, and the closer the distribution of the raised portions 122 in the ordered array structure layer 100 is.

Next, in this embodiment, the ordered array structure layer 120 and the transparent substrate 110 are integrally formed, that is, the ordered array structure layer 120 and the transparent substrate 110 are seamlessly connected, and the ordered array structure layer 120 and the transparent substrate are seamlessly connected. The material of 110 is the same. The material of the ordered array structure layer 120 may be, for example, a transparent polymer material and has a function of resisting biological adhesion.

In this embodiment, the fixed substance 130 is disposed on the raised portions 122 of the ordered array structure layer 120 to capture the cancer cells / bacteria 140. The immobilization substance 130 may include antibodies or other substances suitable for grasping cancer cells / bacteria 140. In this embodiment, the antibody may be, for example, an epithelial cell adhesion molecule (EpCAM) antibody (anti-EpCAM) or a circulating tumor cell antibody. The cancer cell / bacteria 140 may be, for example, a colorectal cancer cell or other circulating tumor cells. And epithelial cell adhesion molecule antibodies or circulating tumor cell antibodies are specific for colorectal cancer cells or other circulating tumor cells. That is, epithelial cell adhesion molecule antibodies or circulating tumor cell antibodies have the ability to identify and capture colorectal cancer cells or other circulating tumor cells.

It should be noted that although the antibody in the optical cancer cell / bacterial detection test strip 100 in this embodiment is an epithelial cell adhesion molecule antibody, the present invention is not limited thereto. In some embodiments, the antibody may also be Yersinia pestis, the cancer cell / bacterium 140 may also be Yersinia pestis, and the Y. pestis antibodies are specific to Yersinia pestis. That is, the Y. pestis antibody has the ability to recognize and capture Y. pestis.

Other embodiments will be listed below for illustration. It must be noted here that the following embodiments use the component numbers and parts of the foregoing embodiments, in which the same reference numerals are used to indicate the same or similar components, and the description of the same technical content is omitted. For the description of the omitted parts, reference may be made to the foregoing embodiments, and the following embodiments are not repeated.

FIG. 2A is a schematic partial perspective view of an optical cancer cell / bacterial detection test strip according to another embodiment of the present invention. FIG. 2B is a schematic cross-sectional view of the optical cancer cell / bacteria detection test strip taken along line B-B 'of FIG. 2A. Please refer to FIG. 1A, FIG. 1B, FIG. 2A, and FIG. 2B at the same time. The optical cancer cell / bacteria test strip 100a of this embodiment is similar to the optical cancer cell / bacteria test strip 100 in FIG. 1A and FIG. 1B, but both The main difference is that in the ordered array structure layer 120a of the optical cancer cell / bacterial detection test strip 100a of this embodiment, the convex portions 122a and the gaps 124a can also be arranged in a two-dimensional array pattern.

In detail, in this embodiment, the convex portion 122a in FIG. 2A is embodied as a columnar protrusion, and the gap 124a is embodied as a hole, wherein the convex portion 122a of the columnar protrusion and the hole-shaped gap 124a They are arranged spaced apart from each other in the first direction X, and the convex portions 122a and the hole-shaped gaps 124a of the columnar protrusions are arranged spaced apart from each other in the second direction Y, and then arranged in a two-dimensional array pattern on the transparent substrate 110.

In this embodiment, please refer to FIG. 2B, the height of the convex portion 122a is H1 ', the height at 1/2 of H1' is H2 ', and the width of the corresponding convex portion 122a at the height H2' is defined as convex. The width W1 'of the rising portion 122a, and the width of the gap 124a corresponding to the height H2' is defined as the pitch G1 'of the gap 124a. The width W1 'of the raised portion 122a is between 100 nm and 10,000 nm. The ratio of the height H1 'to the width W1' of the raised portion 122a is between 0.5 and 5. The ratio of the gap G1 'of the gap 124a to the width W1' of the convex portion 122a is, for example, between 0.2 and 8, preferably, the ratio of the gap G1 'of the gap 124a to the width W1' of the convex portion 122a is, for example, Between 0.5 and 3.

Therefore, in this embodiment, when the width W1 ′ of the raised portion 122a is 500 nm and the working ratio is 0.5, the pitch G1 ′ of the corresponding gap 124a is 250 nm. In other words, approximately 奈 750 nanometers (width W1 '+ pitch G1') will have one raised portion 122a, 毎 1 cm will have 1.3´10 ⁴ raised portions 122a, and an ordered array of 毎 1 cm2 The structure layer 100a will have 1.69´10 ⁸ protrusions 122a. Similarly, when the width W1 'of the raised portion 122a is 500 nm and the working ratio is 8, after conversion, there will be 4.84´10 ⁶ raised portions 122a in the ordered array structure layer 100a of 1 cm2. . In other words, in the ordered array structure layer 100a where the width W1 'of the raised portion 122a is 500 nanometers, since the working ratio is between 0.5 and 8, the density range of the raised portion 122a is between 4.84´10 ⁶ Roots / cm ² to 1.69´10 ⁸ roots / cm ² . It is worth noting that although the width W1 'of the raised portion 122a is 500 nanometers as an example, the present invention is not limited thereto. In other embodiments, the width W1' of the raised portion may be smaller than or Above 500 nm, the calculated density range will also be different.

3A is a schematic partial perspective view of an optical cancer cell / bacterial detection test strip according to another embodiment of the present invention. Fig. 3B is a schematic cross-sectional view of the optical cancer cell / bacteria detection test strip taken along line C-C 'of Fig. 3A. Please refer to FIG. 1A, FIG. 1B, FIG. 3A, and FIG. 3B at the same time. The optical cancer cell / bacterial test strip 200 of this embodiment is similar to the optical cancer cell / bacterial test strip 100 in FIG. 1A and FIG. 1B, but both The main difference is that the optical cancer cell / bacteria detection test strip 200 in this embodiment replaces the transparent substrate 110 of the optical cancer cell / bacteria detection test strip 100 with a high reflectance substrate 250. Here, the material of the high-reflectivity substrate 250 may be, for example, silicon, germanium, gallium arsenide, silicon carbide, indium arsenide, indium phosphide, or other suitable high-reflectivity materials. In some embodiments, the material of the high-reflectivity substrate 250 may also be, for example, a silicon wafer, but is not limited thereto.

In this embodiment, the optical cancer cell / bacterial detection test strip 200 is a reflection-type detection test strip. When the laser light irradiates the optical cancer cell / bacterial detection test sheet 200, the laser light is reflected on the ordered array structure layer 220 of the optical cancer cell / bacterial detection test sheet 200, so that the laser light is diffracted, and the lightning can be measured. The diffraction intensity value is used as an index for detecting cancer cells / bacteria on the surface of the optical cancer cell / bacterial detection test sheet 200. Here, the material of the ordered array structure layer 220 may be, for example, a polymer material having a carboxyl group or an amino group, and the polymer material has a function of resisting biological adhesion. In some embodiments, the material of the ordered array structure layer 220 may also be, for example, polydimethylsiloxane (PDMS), polyethylene terephthalate (PET), or polymethylmethacrylate (PMMA). , But not limited to this.

In this embodiment, the height of the raised portion 222 is H3, and the height at 1/2 H3 is H4. The width of the raised portion 222 corresponding to the height H4 is defined as the width W2 of the raised portion 222, and the height is The width of the gap 224 corresponding to H4 is defined as the gap G2 of the gap 224. The width W2 of the raised portion 122 is between 100 nm and 10,000 nm. The ratio of the height H3 to the width W2 of the raised portion 222 is between 0.5 and 5.

In this embodiment, the ratio of the pitch G2 of the gap 224 to the width W2 of the convex portion 222 is, for example, between 0.2 and 8, preferably, for example, between 0.5 and 3. Therefore, in this embodiment, the ratio obtained by dividing the pitch G2 of the gap 224 by the width W2 of the convex portion 222 is defined as the working ratio of the ordered array structure layer 200. The working ratio may be, for example, between 0.2 and 8, and preferably, the working ratio may be, for example, between 0.5 and 3.

4A is a schematic partial perspective view of an optical cancer cell / bacterial detection test strip according to another embodiment of the present invention. FIG. 4B is a schematic cross-sectional view of the optical cancer cell / bacteria detection test strip taken along line D-D 'of FIG. 4A. Please refer to FIG. 3A, FIG. 3B, FIG. 4A, and FIG. 4B at the same time. The optical cancer cell / bacterial test strip 200a of this embodiment is similar to the optical cancer cell / bacterial test strip 200 in FIG. 3A and FIG. 3B, but both The main difference is that in the ordered array structure layer 220a of the optical cancer cell / bacterial detection test strip 200a of this embodiment, the convex portions 222a and the gaps 224a can also be arranged in a two-dimensional array pattern.

In detail, please refer to FIG. 4A and FIG. 4B, the convex portion 222a may be embodied as a columnar protrusion, and the gap 224a may be embodied as a hole, wherein the columnar convex protrusion 222a and the hole-shaped gap 224a They are arranged spaced apart from each other in the first direction X, and the convex portions 222a of the columnar protrusions and the hole-shaped gaps 224a are arranged spaced apart from each other in the second direction Y, so as to be arranged in a two-dimensional array pattern on the transparent substrate 210.

Please refer to FIG. 4B again, the height of the protruding portion 222a of the ordered array structure layer 220 is H3 ′, and the height at 1/2 of H3 ′ is H4 ′. The width of the corresponding protruding portion 222a at the height H4 ′ is defined Is the width W2 'of the convex portion 222a, and the width of the gap 224a corresponding to the height H4' is defined as the gap G2 'of the gap 224a. The ratio of the pitch G2 'of the gap 224a to the width W2' of the convex portion 222a is, for example, between 0.2 and 8, preferably, the ratio is, for example, between 0.5 and 3. Therefore, the working ratio of the optical cancer cell / bacteria detection test piece 200a of this embodiment may be, for example, between 0.2 and 8, preferably, the working ratio may be, for example, between 0.5 and 3.

5 is a flowchart of a method for detecting an optical cancer cell / bacteria detection test strip according to an embodiment of the present invention. In this embodiment, the detection method of the optical cancer cell / bacterial detection test strip can be used to detect cancer cells / bacteria in the blood.

Referring to FIG. 5, first, step S100 is performed to provide an optical cancer cell / bacterial detection test strip and a sample obtained from blood. In this embodiment, the optical cancer cell / bacterial detection test strips are the above-mentioned penetrating detection test strips 100 and 100a or reflection type detection test strips 200 and 200a. The source of the sample is blood. The method for obtaining the sample is, for example, density centrifugation (for example, Ficoll), that is, the blood is first centrifuged to separate red blood cells, white blood cells, platelets, plasma, and cancer cells / bacteria from the blood 140, 240. And other substances. Next, a buffy coat after centrifugation of blood is taken as a sample, wherein the brown yellow layer includes white blood cells and cancer cells / bacteria 140, 240.

Next, step S110 is performed to drop the sample on the optical cancer cell / bacterial detection test strip, and react the sample with the fixed substance of the optical cancer cell / bacterial detection test strip to obtain a reaction optical cancer cell / bacterial detection test strip. In this embodiment, the taken out yellow-brown layer is directly dropped on the optical cancer cell / bacterial detection test strips 100, 100a, 200, 200a, and left for a period of time to allow the cells in the yellow-brown layer to settle and interact with the optical cancer The fixed substances 130 and 230 in the cell / bacterial detection test strips 100, 100a, 200, and 200a react. At this time, the fixed substances 130 and 230 in the optical cancer cell / bacteria detection test strips 100, 100a, 200, and 200a can recognize and grasp the cancer cells / bacteria 140 and 240 in the brown-yellow layer, but will not grasp the brown-yellow White blood cells in layers. Therefore, after the washing solution is added, other cells in the sample that have not reacted with the fixed substances 130 and 230 can be removed. In other words, on the optical cancer cell / bacterial detection test strips 100, 100a, 200, and 200a that are resistant to biological adhesion, the white blood cells in the sample will not be grasped by the fixed substances 130, 230 and will not stick to the optical cancer. The cell / bacteria detection test strips 100, 100a, 200, 200a are, therefore, the white blood cells on the optical cancer cell / bacteria detection test strips 100, 100a, 200, 200a can be removed after the washing solution is added.

It should be noted that the fixing substances 130 and 230 may be, for example, antibodies or other materials that can specifically identify and capture cancer cells / bacteria 140 and 240 in the blood. For example, the fixing substances 130 and 230 may be epithelial cell adhesion Molecular antibodies are used to capture colorectal cancer cells or other circulating tumor cells in the blood, but the present invention is not limited thereto. In some embodiments, the immobilizing substance may be a cancer cell / Yersinia antibody to capture cancer cells / Yersinia in the blood.

Next, step S120 is performed to provide laser light, so that the laser light irradiates the optical cancer cell / bacteria detection test strip after the reaction, and detects the laser diffraction intensity value after the laser light is diffracted. In this embodiment, a laser analyzer is used to provide laser light, and the laser light is irradiated on the optical cancer cell / bacterial detection test strips 100, 100a, 200, and 200a after reacting with the sample. At this time, since the optical cancer cell / bacterial detection test strips 100, 100a, 200, and 200a have the ordered array structure layers 120, 120a, 220, and 220a, the laser light can be diffracted. Next, a laser analyzer is used to detect and quantify the intensity of the laser light after it is diffracted to obtain the laser diffraction intensity value.

Next, step S130 is performed, and the laser diffraction intensity value measured by the optical cancer cell / bacteria detection test strip after the reaction is compared with the laser diffraction measured by the optical cancer cell / bacteria detection test strip before the sample is added. Compared with the intensity value, when the laser diffraction intensity value measured by the optical cancer cell / bacterial test strip after the reaction decreases, it means that the cancer cells / bacteria are contained in the blood. In this embodiment, the laser diffraction intensity values of the optical cancer cells / bacteria detection test strips 100, 100a, 200, and 200a before the sample is added to the detection side by a laser analyzer, and then the reaction optical The laser diffraction intensity values of the cancer cell / bacterial detection test strips 100, 100a, 200, and 200a were compared. At this time, the circulating tumor cells or Yersinia pestis captured on the surface of the ordered array structure layer 120, 120a, 220, 220a will destroy the surface pattern of the ordered array structure layer 120, 120a, 220, 220a, that is, the damage The one-dimensional periodic pattern or two-dimensional array pattern in which the raised portions 122, 122a, 222, and 222a and the gaps 124, 124a, 224, and 224a are neatly arranged, thereby reducing the laser diffraction intensity value. In other words, when the laser diffraction intensity value decreases from the detection side, it means that the detected blood contains cancer cells / bacteria 140, 240. Therefore, in this embodiment, the laser diffraction intensity value can be used as an index for detecting the cancer cells / bacteria 140 and 240 on the surfaces of the optical cancer cell / bacterial detection test strips 100, 100a, 200, and 200a.

[Experimental example]

[Experimental Example 1] Detection of circulating tumor cells in the blood of healthy subjects and the blood of cancer patients.

In this embodiment, the blood of a patient with colorectal cancer is taken as an example, but the present invention is not limited thereto. In other embodiments, the optical cancer cell / bacterial detection test strip and the detection method of the present invention can also be used to detect the blood of patients with breast cancer or the blood of patients with oral cancer. That is, in some embodiments, the circulating tumor cells that can also be detected by the optical cancer cell / bacterial detection test strip and the detection method thereof include colorectal cancer cells, breast cancer cells, or oral cancer cells.

According to the above detection method, first, centrifuge 3 ml of blood from healthy subjects and 3 ml of colorectal cancer patients, and then take the brown-yellow layer as a sample of healthy subjects (referred to as healthy Samples) and colorectal cancer patients (referred to as colorectal cancer samples). Among them, the brown-yellow layer taken from 3 ml of blood includes about 2 × 10 ⁸ or more white blood cells.

Next, the healthy sample and the colorectal cancer sample are dropped on different penetrating detection test strips 100a, so that the healthy sample reacts with the fixed substance 130 of the optical cancer cell / bacterial detection test strip 100a, and the colorectal cancer sample and another An inspection of the fixed substance 130 of the optical cancer cell / bacterial test strip 100a is performed. Then, after washing liquid is added and other cells in the sample that have not reacted with the immobilized substance 130 are removed, the reacted optical cancer cell / bacterial test strip 100a is irradiated with laser light. Then, a laser analyzer is used to detect the side and quantify the laser diffraction intensity of the optical cancer cell / bacterial test strip 100a after the laser light penetrating reaction to obtain the laser diffraction intensity value.

FIG. 6A is a laser diffraction intensity value of a sample from different sources after reacting with an optical cancer cell / bacteria test strip in an experimental example of the present invention. Please refer to FIG. 6A. The laser diffraction intensity value before adding a sample (referred to as the control group) is about 3.4 × 10 ⁷ cnts, the laser diffraction intensity value of a healthy sample is about 3.4 × 10 ⁷ cnts, and the colorectal cancer sample. The laser diffraction intensity value is about 3.1 × 10 ⁷ cnts. Among them, since the laser diffraction intensity value of the healthy sample is similar to the laser diffraction intensity value of the control group, it means that there are no cancer cells / bacteria in the healthy sample, so the surface pattern of the ordered array structure layer 120a and the lightning will not be affected. The value of the diffraction intensity affects. However, compared with the laser diffraction intensity value of the control group or the laser diffraction intensity value of the healthy sample, the laser diffraction intensity value of the colorectal cancer sample decreased significantly. Therefore, it means that the colorectal cancer sample contains cancer cells / The bacteria 140 thus destroy the surface patterns of the ordered array structure layers 120 and 120a and cause the laser diffraction intensity value to decrease significantly.

[Experimental Example 2] Circulating tumor cells on an optical cancer cell / bacterial test strip were cultured, and the number of cells was measured by laser.

Taking colorectal cancer cells as an example, about 500 colorectal cancer cells were added to the penetrating detection test strip 100 or the penetrating detection test strip 100a and cultured, and the culture was performed on the first, fourth, seventh, and tenth days after the culture. The number of cells was measured by fluorescent staining and laser irradiation. Among them, the control group is an optical cancer cell / bacterial detection test sheet 100 without adding colorectal cancer cells.

[Table 1] Results of colorectal cancer cells cultured on optical cancer cell / bacterial test strip 100 Days after culture Number of cells after fluorescent staining Laser diffraction intensity value Control group 0 4.78 × 10 ⁷ cnts Day 1 457 4.44 × 10 ⁷ cnts Day 4 912 3.57 × 10 ⁷ cnts Day 7 5236 2.49 × 10 ⁷ cnts Day 10 50000 1.16 × 10 ⁷ cnts

According to the results in Table 1, the laser diffraction intensity value of the control group is the highest. Next, after colorectal cancer cells are added to the optical cancer cell / bacteria detection test strip 100 and cultured, as the number of days of culture increases, the number of cells after fluorescent staining also relatively increases. Because the increased cells will damage the surface pattern of the ordered array structure layer 120, the laser diffraction intensity value will decrease as the number of cells increases. In other words, in this embodiment, compared with the fluorescent staining method, the measurement method using laser can also be used to indicate the change in the number of reaction cells.

[Table 2] Results of colorectal cancer cells cultured on optical cancer cell / bacterial test strip 100a Days after culture Number of cells after fluorescent staining Laser diffraction intensity value Control group 0 4.53 × 10 ⁷ cnts Day 1 413 4.05 × 10 ⁷ cnts Day 4 783 3.52 × 10 ⁷ cnts Day 7 4547 2.62 × 10 ⁷ cnts Day 10 43333 1.14 × 10 ⁷ cnts

According to the results in Table 2, it can be seen that the laser diffraction intensity value of the control group 100a is the highest. Next, after adding colorectal cancer cells to the optical cancer cell / bacterial test strip 100a and culturing them, as the number of days of culture increases, the number of cells after fluorescent staining also relatively increases, and because the increased cells will orderly The surface pattern of the array structure layer 120a causes damage, which further causes the laser diffraction intensity value to decrease as the number of cells increases. In other words, in this embodiment, compared with the fluorescent staining method, the measurement method using laser can also be used to indicate the change in the number of reaction cells.

It is worth noting that the method of fluorescent staining often takes several hours to know the results, and the cells after fluorescent staining can no longer be cultured. However, the laser measurement method can be used to know the change in cell number immediately, and the measured cells can still be cultured. Therefore, compared with the conventional detection method, the optical cancer cell / bacterial detection test strip 100 and the detection method of the embodiment have the effect of less sample demand and rapid detection.

In addition, in addition to the optical cancer cell / bacterial detection test strip 100 and the detection method of this embodiment, in addition to detecting circulating tumor cells in blood, when the circulating tumor cells in the blood are captured in the optical cancer cell / bacterial detection test After the tablets 100 and 100a are cultured, the cultured circulating tumor cells can also be provided for clinical application, and even used as follow-up research on personalized medicine.

[Experimental Example 3] Detection of Yersinia pestis in blood

In the present embodiment, were added different amounts (10 ² to 10 ⁷⁾ pestis or different concentrations (10 ² to 10 ⁷⁾ Escherichia coli in healthy human blood, and the reflecting type detection test The sheet 200 is detected according to the above-mentioned detection method.

First, 3 ml of blood containing Yersinia pestis and 3 ml of blood containing E. coli were centrifuged, and then the brown-yellow layer was taken out as a sample containing Y. pestis (referred to as Y. pestis sample) and a sample containing E. coli (referred to as For E. coli samples). Among them, the brown-yellow layer taken from 3 ml of blood includes about 2 × 10 ⁸ or more white blood cells.

Next, the Y. pestis sample and the E. coli sample are dropped on different optical cancer cell / bacterial detection test strips 200, respectively, so that the Y. pestis sample and the E. coli sample react with the fixed substance 230, respectively. Here, the immobilized substance 230 is a Yersinia pestis antibody. Then, after adding the washing solution and removing other cells in the sample that have not reacted with the immobilized substance 230, the reacted optical cancer cell / bacterial test strip 200 is irradiated with laser light. Then, a laser analyzer is used to detect the side and quantify the laser diffraction intensity reflected by the laser light on the reacted optical cancer cell / bacteria test strip 200 to obtain the laser diffraction intensity value. The detection result is as follows: Figure 6B.

FIG. 6B is a laser diffraction intensity value of a sample from different sources after being reacted with an optical cancer cell / bacteria test strip in another experimental example of the present invention. Please refer to FIG. 6B. In the E. coli sample, as the number of E. coli increases, the logarithms of the laser diffraction intensity values after the reaction are all between 7.6 and 7.7. That is, even if the number of samples in E. coli increases of from 10 ² to 10 ^7, an optical cancer cells / bacteria detection test strip 200 is fixed to the material 230 still not crawl E. coli, such that an ordered array The surface pattern of the structure layer 220 is not damaged, so that the laser diffraction intensity of the reflected laser light is maintained at a similar value. In contrast, in the Y. pestis samples, as the number of Y. pestis increased, the logarithm of the laser diffraction intensity value after the reaction decreased from about 7.7 to about 7.3. That is, when the number of samples in Y. pestis pestis increase of from 10 ² to 10 ^7, an optical cancer cells / bacteria test strip 100 is fixed to the material 230 crawls more pestis, such that grasping The picked Yersinia pestis destroys the surface pattern of the ordered array structure layer 220, thereby causing the laser diffraction intensity of the reflected laser light to decrease.

It should be noted that, shown in Figure 6B, when the number of Yersinia pestis at from 10 ² to 10 ^7, and the logarithm of the number of laser diffraction pestis logarithmic intensity values exhibit a negative correlation, the correlation The coefficient R ² is 0.9679 and the corresponding equation is Y = -0.0762X + 7.8412. Among them, Y is the logarithm of the laser diffraction intensity value, and X is the logarithm of the number of Yersinia pestis. Therefore, in clinical testing, when the logarithm of the laser diffraction intensity value after detection falls between 7.3 and 7.7, the above equation can be used to calculate the amount of Yersinia pestis in the blood.

In summary, in the optical cancer cell / bacterial detection test strip and the detection method of the present invention, the optical cancer cell / bacterial detection test strip includes an ordered array structure layer and a fixed substance capable of grasping the cancer cells / bacteria. The width of the raised portion is between 100 nm and 10000 nm, and the ratio of the height to the width of the raised portion is between 0.5 and 5. The ratio of the gap distance to the width of the raised portion is between Between 0.2 and 8. Therefore, the laser light can be used to detect the optical cancer cell / bacteria detection test strip after reacting with the blood sample. When the laser diffraction intensity value measured by the reacted optical cancer cell / bacterial detection strip decreases, This means that the blood sample contains cancer cells / bacteria. With this design, the optical cancer cell / bacterial detection test strip provided by the present invention has the advantages of simple manufacturing process and low price. In addition, the method for detecting an optical cancer cell / bacteria test strip provided by the present invention can be used to detect cancer cells / bacteria in blood, and has the advantages of less sample demand and rapid detection.

Although the present invention has been disclosed as above with the examples, it is not intended to limit the present invention. Any person with ordinary knowledge in the technical field can make some modifications and retouching without departing from the spirit and scope of the present invention. The protection scope of the present invention shall be determined by the scope of the attached patent application.

100, 100a, 200, 200a ‧‧‧ Optical Cancer Cell / Bacteria Test Strips

110‧‧‧ transparent substrate

120, 120a, 220, 220a‧‧‧ ordered array structure layer

122, 122a, 222, 222a‧‧‧ raised

124, 124a, 224, 224a

130, 230‧‧‧ fixed substances

140, 240‧‧‧ cancer cells / bacteria

250‧‧‧High reflectivity substrate

G1, G1 ’, G2, G2’ ‧‧‧ pitch

H1, H1 ’, H2, H2’, H3, H3 ’, H4, H4’ ‧‧‧ height

S100, S110, S120, S130 ‧‧‧ steps

W1, W1 ’, W2, W2’‧‧‧Width

FIG. 1A is a schematic partial perspective view of an optical cancer cell / bacterial detection test strip according to an embodiment of the present invention. Fig. 1B is a schematic cross-sectional view of the optical cancer cell / bacteria detection test strip taken along line A-A 'of Fig. 1A. FIG. 2A is a schematic partial perspective view of an optical cancer cell / bacterial detection test strip according to another embodiment of the present invention. FIG. 2B is a schematic cross-sectional view of the optical cancer cell / bacteria detection test strip taken along line B-B 'of FIG. 2A. 3A is a schematic partial perspective view of an optical cancer cell / bacterial detection test strip according to another embodiment of the present invention. Fig. 3B is a schematic cross-sectional view of the optical cancer cell / bacteria detection test strip taken along line C-C 'of Fig. 3A. 4A is a schematic partial perspective view of an optical cancer cell / bacterial detection test strip according to another embodiment of the present invention. FIG. 4B is a schematic cross-sectional view of the optical cancer cell / bacteria detection test strip taken along line D-D 'of FIG. 4A. 5 is a flowchart of a method for detecting an optical cancer cell / bacteria detection test strip according to an embodiment of the present invention. FIG. 6A is a laser diffraction intensity value of a sample from different sources after reacting with an optical cancer cell / bacteria test strip in an experimental example of the present invention. FIG. 6B is a laser diffraction intensity value of a sample from different sources after being reacted with an optical cancer cell / bacteria test strip in another experimental example of the present invention.

Claims (14)

An optical cancer cell / bacterial detection test strip includes: a high reflectance / transparent substrate; an ordered array structure layer disposed on the high reflectance / transparent substrate, the ordered array structure layer includes: a plurality of protrusions And a plurality of gaps between the raised portions, wherein a width of each of the raised portions is between 100 nm and 10,000 nm, and a ratio of a height of each raised portion to the width Between 0.5 to 5, the ratio of a distance between each of the gaps to the width of each of the protrusions is between 0.2 and 8; and a plurality of fixed substances arranged in the ordered array structure layer. The convex portion is used to grasp the cancer cells / bacteria. When a laser light irradiates the optical cancer cell / bacteria test strip, the laser light is diffracted and a laser diffraction intensity value is obtained. It serves as an index for detecting cancer cells / bacteria on the surface of the optical cancer cell / bacterial detection test strip. The optical cancer cell / bacterial detection test strip according to item 1 of the scope of the patent application, wherein the optical cancer cell / bacterial detection test strip is a penetrating detection test strip, and the manner of diffracting the laser light includes making the laser The transmitted light penetrates the ordered array structure layer of the optical cancer cell / bacterial detection test strip. According to the optical cancer cell / bacterial detection test strip described in the first item of the patent application scope, the protrusions and the gaps are arranged in a one-dimensional periodic pattern or a two-dimensional array pattern. The optical cancer cell / bacterial detection test strip according to item 3 of the patent application scope, wherein the one-dimensional periodic pattern includes a line shape and a groove. The optical cancer cell / bacterial detection test strip according to item 3 of the patent application scope, wherein the two-dimensional array pattern includes a pillar shape and a hole. The optical cancer cell / bacterial detection test strip according to item 1 of the scope of the patent application, wherein the material of the transparent substrate includes polydimethylsiloxane. The optical cancer cell / bacteria detection test strip according to item 1 of the patent application scope, wherein the optical cancer cell / bacteria detection test strip is a reflection detection test strip, and the manner of diffracting the laser light includes making the laser light Reflected on the ordered array structure layer of the optical cancer cell / bacterial detection test strip. The optical cancer cell / bacterial detection test strip as described in the first item of the patent application scope, wherein the material of the high reflectance substrate includes a silicon wafer. The optical cancer cell / bacterial detection test strip as described in item 1 of the patent application scope, wherein the fixed substances include antibodies. The optical cancer cell / bacteria test strip according to item 1 of the scope of the patent application, wherein the cancer cells / bacteria include circulating tumor cells or Yersinia pestis. A method for detecting an optical cancer cell / bacterial detection test strip includes: providing the optical cancer cell / bacterial detection test strip as described in any one of items 1 to 10 of the patent application scope and a sample; dropping the sample On the optical cancer cell / bacterial detection test strip, and making the sample react with the fixed substances of the optical cancer cell / bacterial detection test strip to obtain the reacted optical cancer cell / bacterial detection test strip; provide the thunder Irradiate the laser light with the optical cancer cell / bacteria test strip after the reaction, and detect the laser diffraction intensity value after the laser light is diffracted; The laser diffraction intensity value measured by the test piece is compared with the laser diffraction intensity value measured by the optical cancer cell / bacteria detection test strip before the sample is added. When the laser diffraction intensity value measured by the cell / bacteria test strip decreases, it means that the cancer cells / bacteria are contained in the blood. The method for detecting an optical cancer cell / bacterial detection test strip according to item 11 of the patent application scope, wherein the laser light is diffracted in a manner that allows the laser light to penetrate the optical cancer cell / bacterial detection test strip. The ordered array structure layer, or the laser light is reflected on the ordered array structure layer of the optical cancer cell / bacterial detection test strip. The method for detecting an optical cancer cell / bacteria detection test strip according to item 11 of the scope of patent application, wherein the method for obtaining the sample comprises: centrifuging a blood; and taking out a brown-yellow layer. The method for detecting an optical cancer cell / bacterial detection test strip according to item 11 of the patent application scope, wherein before providing the laser light, it further comprises adding a washing solution to remove cells in the sample that have not reacted with the fixed substances .