TWI600906B - Composite poly fiber slide and usage thereof - Google Patents
Composite poly fiber slide and usage thereof Download PDFInfo
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Description
本發明係關於一種高分子纖維癌症檢測試片，尤其是一種利用在循環腫瘤細胞篩檢的高分子複合纖維薄膜。 The invention relates to a polymer fiber cancer test piece, in particular to a polymer composite fiber film which is used for screening of circulating tumor cells.
癌症為目前居高不下的死亡原因之一，癌症細胞自病灶轉移至身體其他部位，是其中常見的症狀。癌症細胞轉移主要來自循環腫瘤細胞在體內流動，因此如何偵測血液中循環腫瘤細胞的數量或遷移方向，可以作為癌症轉移的判斷依據。但是在癌症發展初期，循環腫瘤細胞數目相對較少，患者體內僅白血球數目增加，在患有轉移性癌症之患者中，循環腫瘤細胞為稀有細胞，大約每109個寫細胞有一個循環腫瘤細胞，因此自血液中檢測循環腫瘤細胞需透過集中收集、分離等等，檢測程序相當複雜。以大腸癌為例，習知診斷大腸癌方式主要透過內視鏡檢測法。雖然內視鏡病理檢測具有高準確性，但需要醫師執行內視鏡手術再透過病理師處理切片並分析，檢測過程需要多方人力且耗時。另外，內視鏡檢測為侵入式檢測，對患者造成其他不便。 Cancer is one of the leading causes of death, and cancer cells are often transmitted from the lesion to other parts of the body. Cancer cell metastasis mainly comes from the circulation of circulating tumor cells in the body, so how to detect the number or migration direction of circulating tumor cells in the blood can be used as a basis for judging cancer metastasis. However, in the early stage of cancer development, the number of circulating tumor cells is relatively small, and the number of white blood cells is increased in patients. In patients with metastatic cancer, circulating tumor cells are rare cells, and there is one circulating tumor cell per 10 9 writing cells. Therefore, the detection of circulating tumor cells from the blood requires centralized collection, separation, etc., and the detection procedure is quite complicated. Taking colorectal cancer as an example, the conventional method for diagnosing colorectal cancer is mainly through endoscopic detection. Although endoscopic pathology has high accuracy, it requires a physician to perform endoscopic surgery and then process the slice through the pathologist and analyze it. The detection process requires multiple labors and is time consuming. In addition, the endoscope detection is an invasive detection, causing other inconvenience to the patient.
目前急需一種非侵入式，且可快速、有效檢測癌症以及循環腫瘤細胞的方法，排除其他循環血細胞之非特異性結合，同時可免去目前癌症檢測所需的大量人力、縮短診斷時間。 There is an urgent need for a non-invasive, rapid and effective method for detecting cancer and circulating tumor cells, eliminating non-specific binding of other circulating blood cells, while eliminating the need for a large amount of manpower required for cancer detection and shortening the diagnosis time.
鑑於上述課題，本發明旨在提供一種高分子纖維癌症檢測試片包含一薄膜以及複數個具有選擇性之生物活性組成連接於該薄膜，該薄膜係以一聚合物包含聚乙二醇以及一與聚乙二醇具有親和性之高分子，該聚合物以抽絲形成片狀薄膜。 In view of the above problems, the present invention is directed to a polymer fiber cancer test strip comprising a film and a plurality of selective bioactive components attached to the film, the film comprising a polymer comprising polyethylene glycol and a Polyethylene glycol has an affinity polymer which is drawn into a sheet-like film by spinning.
根據本發明部份實施例，該與聚乙二醇具有親和性之高分子為非水溶性。 According to some embodiments of the present invention, the polymer having affinity with polyethylene glycol is water-insoluble.
根據本發明部份實施例，該生物活性組成對於循環腫瘤細胞具有專一性。 According to some embodiments of the invention, the bioactive composition is specific for circulating tumor cells.
本發明還提供一種高分子纖維癌症檢測試片作為篩檢特定細胞之用途，該用途包含製備一血液樣本，培養該血液樣本所含之細胞，塗覆該血液樣本所含之細胞於一高分子纖維癌症檢測試片，以及透過螢光顯影篩檢該高分子纖維癌症檢測試片上之一特定細胞。該高分子纖維癌症檢測試片包含一薄膜以及複數個具有選擇性之生物活性組成連接於該薄膜，該薄膜係以一聚合物包含聚乙二醇以及一與聚乙二醇具有親和性之高分子，該聚合物以抽絲形成片狀薄膜。 The present invention also provides a polymer fiber cancer test piece for screening a specific cell, the use comprising preparing a blood sample, culturing the cell contained in the blood sample, and coating the cell contained in the blood sample in a polymer A fiber cancer test strip, and a specific cell on the polymer fiber cancer test strip is screened by fluorescence development. The polymer fiber cancer test strip comprises a film and a plurality of selective bioactive components attached to the film, the film comprising a polymer comprising polyethylene glycol and having a high affinity with polyethylene glycol. Molecules, the polymer is drawn into a sheet-like film by spinning.
本發明之試片材料取得容易，可大量生產，且透過改質後抓取循環腫瘤細胞，同時其粗糙表面減少非特定 白血球沾黏，降低干擾判斷實驗結果的因素。另外，此試片以非侵入式方法篩檢循環腫瘤細胞，減少相關人力成本，縮短實驗時間。 The test piece material of the invention is easy to obtain, can be mass-produced, and the circulating tumor cells are grasped after being modified, and the rough surface thereof is reduced to be non-specific. White blood cells are sticky, reducing the factors that interfere with the experimental results. In addition, this test piece screens circulating tumor cells in a non-invasive manner, reducing the associated labor costs and shortening the experimental time.
10‧‧‧高分子纖維癌症檢測試片 10‧‧‧ Polymer Fiber Cancer Test Strips
100‧‧‧薄膜 100‧‧‧film
120‧‧‧連結分子 120‧‧‧Linking molecules
130‧‧‧生物活性組成 130‧‧‧Bioactive composition
本發明之上述和其他態樣、特徵及其他優點參照說明書內容並配合附加圖式得到更清楚的了解，其中： The above and other aspects, features, and other advantages of the present invention will be more clearly understood by reference to the description and the accompanying drawings.
〔第1圖〕繪示係依照本發明之一實施方式的一種高分子纖維癌症檢測試片示意圖。 [Fig. 1] is a schematic view showing a polymer fiber cancer test piece according to an embodiment of the present invention.
〔第2圖〕係依照本發明之一實施方式的一種高分子纖維癌症檢測試片之薄膜的電子顯微鏡圖。 [Fig. 2] An electron micrograph of a film of a polymer fiber cancer test piece according to an embodiment of the present invention.
〔第3A-3D圖〕係依照本發明之一實施方式的一種高分子纖維癌症檢測試片之薄膜無水以及浸水後的電子顯微鏡圖。 [Fig. 3A-3D] is an electron micrograph of a film of a polymer fiber cancer test piece according to an embodiment of the present invention, which is anhydrous and water-immersed.
〔第4A-4D圖〕係依照本發明之一實施方式的一種高分子纖維癌症檢測試片之薄膜無水以及浸水後的電子顯微鏡圖。 [Fig. 4A-4D] is an electron micrograph of a film of a polymer fiber cancer test piece according to an embodiment of the present invention, which is anhydrous and water-immersed.
〔第5圖〕係依照本發明之一實施方式的一種高分子纖維癌症檢測試片吸附之循環腫瘤細胞顆粒數直方圖。 [Fig. 5] A histogram of the number of circulating tumor cell particles adsorbed by a polymer fiber cancer detecting test piece according to an embodiment of the present invention.
〔第6圖〕係依照本發明之一實施方式的一種高分子纖維癌症檢測試片吸附之細胞暗視野螢光顯影圖。 [Fig. 6] A dark-field fluorescence development image of a cell adsorbed on a polymer fiber cancer test piece according to an embodiment of the present invention.
〔第7圖〕係依照本發明之一實施方式的一種高分子纖維癌症檢測試片吸附之細胞亮視野螢光顯影圖。 [Fig. 7] Fig. 7 is a bright-field fluorescence development image of a cell adsorbed by a polymer fiber cancer detecting test piece according to an embodiment of the present invention.
為了使本揭示內容的敘述更加詳盡與完備，下文針對了本發明的實施態樣與具體實施例提出了說明性的描述；但這並非實施或運用本發明具體實施例的唯一形式。以下所揭露的各實施例，在有益的情形下可相互組合或取代，也可在一實施例中附加其他的實施例，而無須進一步的記載或說明。在以下描述中，將詳細敘述許多特定細節以使讀者能夠充分理解以下的實施例。然而，可在無此等特定細節之情況下實踐本發明之實施例。 The description of the embodiments of the present invention is intended to be illustrative and not restrictive. The embodiments disclosed herein may be combined or substituted with each other in an advantageous manner, and other embodiments may be added to an embodiment without further description or description. In the following description, numerous specific details are set forth However, embodiments of the invention may be practiced without these specific details.
請參考第1圖。本發明係關於一種高分子纖維癌症檢測高分子纖維癌症檢測試片10包含一薄膜100以及複數個具有選擇性之生物活性組成130連接於該薄膜100。該高分子纖維癌症檢測試片10可有效捕捉循環腫瘤細胞〈Circulating tumor cells,CTCs〉或其類似物〈例如：細菌、病毒、內皮細胞〉。 Please refer to Figure 1. The present invention relates to a polymer fiber cancer detecting polymer fiber cancer detecting test piece 10 comprising a film 100 and a plurality of selective bioactive components 130 attached to the film 100. The polymer fiber cancer test strip 10 can effectively capture circulating tumor cells (CTCs) or analogs thereof (eg, bacteria, viruses, endothelial cells).
根據本發明部份實施例，該薄膜100可以由聚乙二醇〈poly(ethylene glycol),PEG〉、〈poly(ethylene oxide),PEO〉以及一與聚乙二醇具有親和性之高分子形成一聚合物。聚乙二醇可減少非特定細胞結合及血清蛋白吸附，聚乙二醇較佳具有約1000至30萬分子量之間。然而，聚乙二醇為水溶性，無法單獨成為固化纖維，遇到血清裡的高水含量產生水解。請參考第3A圖，此薄膜僅由聚乙二醇纖維組成，遇水之後，產物發生水解，請參考第3B圖，完全失去乾糙時的態樣，原本明顯絲狀的外型已完全變形。因此需添加非水溶性且同時與聚乙二醇相溶之高分子形成一 聚合物，該聚合物經過紡織程序後具有固定形狀。根據本發明一實施例，聚乙二醇以及與聚乙二醇具有親和性之高分子之混和重量比率介於0.2-0.9之間。更高的聚乙二醇含量雖可提高抗生物沾黏的功效，但同時降低欲捕捉的特定細胞結合能力，且織物水溶性提高，可能造成高分子纖維癌症檢測試片10碰到血清時水解的現象，影響高分子纖維癌症檢測試片10的完整性。請參考第3C圖。此為90%聚乙二醇與10%與聚乙二醇相溶之高分子之產物。在織物上加水之後，請參考第3D圖，水解的現象雖較為緩和，但織物的外型仍嚴重走樣。請參考第4A圖，此為80%聚乙二醇與20%與聚乙二醇相溶之高分子之產物在無水的狀態。加水之後，請參考第4B圖，已無法分辨織物纖維外型。過低的聚乙二醇含量使得薄膜100抗生物沾黏功能降低，將會吸引非特定細胞附著，則失去高分子纖維癌症檢測試片10對於特定細胞的專一性。 According to some embodiments of the present invention, the film 100 may be formed of polyethylene glycol (poly(ethylene glycol), PEG>, <poly(ethylene oxide), PEO, and a polymer having affinity with polyethylene glycol. a polymer. Polyethylene glycol can reduce non-specific cell binding and serum protein adsorption, and polyethylene glycol preferably has a molecular weight of between about 1,000 and 300,000. However, polyethylene glycol is water-soluble and cannot be cured alone. It is hydrolyzed by high water content in serum. Please refer to Figure 3A. This film consists only of polyethylene glycol fiber. After the water is encountered, the product is hydrolyzed. Please refer to Figure 3B. The pattern is completely lost when dry and rough. The original filament-like shape has been completely deformed. . Therefore, it is necessary to add a polymer which is water-insoluble and which is compatible with polyethylene glycol at the same time. A polymer that has a fixed shape after a textile process. According to an embodiment of the present invention, the blended weight ratio of polyethylene glycol and a polymer having affinity with polyethylene glycol is between 0.2 and 0.9. Higher polyethylene glycol content can improve the anti-bioadhesive effect, but at the same time reduce the specific cell binding ability to be captured, and the water solubility of the fabric is increased, which may cause the polymer fiber cancer test strip 10 to hydrolyze when it hits serum. The phenomenon affects the integrity of the polymer fiber cancer test strip 10. Please refer to Figure 3C. This is the product of a polymer of 90% polyethylene glycol and 10% polyethylene glycol. After adding water to the fabric, please refer to the 3D figure. Although the hydrolysis phenomenon is mild, the appearance of the fabric is still seriously distorted. Please refer to Fig. 4A, which is a product of 80% polyethylene glycol and 20% polyethylene glycol-soluble polymer in a water-free state. After adding water, please refer to Figure 4B, it is impossible to distinguish the fabric fiber appearance. The too low polyethylene glycol content causes the anti-bioadhesive function of the film 100 to be lowered, which will attract non-specific cell adhesion, and the specificity of the polymer fiber cancer test strip 10 for a specific cell is lost.
薄膜100係先透過電紡絲、抽絲或其它方式形成由聚乙二醇以及該與聚乙二醇具有親和性之高分子之單絲，再透過無方向性的紡織方式形成片狀薄膜100。透過電紡絲的方式可製作出表面積大的單股絲。由聚乙二醇以及與聚乙二醇具有親和性之高分子之單股絲之一直徑50奈米〈nm〉至2微米〈μm〉。請參考第2圖。片狀薄膜100之孔隙介於10奈米〈nm〉至30微米〈μm〉，而一般細胞大小約為10微米，因此薄膜100可有效捕捉細胞，使得細胞滯留於其上。 The film 100 is formed by electrospinning, spinning or other means to form a monofilament of polyethylene glycol and the polymer having affinity with polyethylene glycol, and then forming a sheet-like film 100 by a non-directional textile method. . A single strand of wire having a large surface area can be produced by electrospinning. One of the single-strand filaments of polyethylene glycol and a polymer having affinity with polyethylene glycol has a diameter of 50 nm <nm> to 2 μm <μm>. Please refer to Figure 2. The pores of the sheet-like film 100 are between 10 nm < nm> and 30 μm < μm>, and the general cell size is about 10 μm, so that the film 100 can effectively capture cells and cause cells to stay thereon.
與聚乙二醇具有親和性之高分子包含例如聚醯胺、聚氨酯、甲殼素〈chitosan〉或其它合適的高分子。根據本發明一實施例，與聚乙二醇具有親和性之高分子係指尼龍6〈nylon 6〉。聚乙二醇以及尼龍6以30/70之重量百分比率混和，將聚乙二醇以及尼龍6直接加入70%甲酸〈HCOOH〉攪拌後溶解，接著透過電紡絲的方式成絲，電紡絲的操作條件包含操作電壓約為15伏〈V〉，工作距離約15公分〈cm〉，抽絲的流速約0.5毫升/小時〈ml/hr〉，以形成片狀薄膜100。請參考第4C圖，此為由聚乙二醇以及尼龍6以30/70之重量百分比率混和產生之薄膜100在無水的狀態。請參考第4D圖，加水之後，薄膜100之單股絲仍清晰可辨，與無水狀態之態樣並無太大差異。表示該聚合物同時具備聚乙二醇的抗生物沾黏優勢，且不容易水解。薄膜100之纖維穩定性大為提升。纖維製備方式並不侷限於電紡絲，其它可以製成纖維化高分子的手段也可運用製成薄膜。 The polymer having affinity with polyethylene glycol includes, for example, polyamine, polyurethane, chitin or other suitable polymer. According to an embodiment of the present invention, the polymer having affinity with polyethylene glycol means nylon 6 <nylon 6>. Polyethylene glycol and nylon 6 were mixed at a weight ratio of 30/70, and polyethylene glycol and nylon 6 were directly added to 70% formic acid <HCOOH>, dissolved, and then spun by electrospinning, electrospinning. The operating conditions include an operating voltage of about 15 volts <V>, a working distance of about 15 cm < cm>, and a spinning flow rate of about 0.5 ml/hr <ml/hr> to form a sheet-like film 100. Please refer to FIG. 4C, which is a state in which the film 100 produced by mixing polyethylene glycol and nylon 6 at a weight ratio of 30/70 is in a water-free state. Please refer to Figure 4D. After adding water, the single strand of the film 100 is still clearly distinguishable, and there is not much difference from the state of the anhydrous state. It indicates that the polymer has the anti-bioadhesive advantage of polyethylene glycol and is not easily hydrolyzed. The fiber stability of the film 100 is greatly improved. The fiber preparation method is not limited to electrospinning, and other means for forming a fiberized polymer can also be used as a film.
由聚乙二醇以及與聚乙二醇具有親和性之高分子形成之聚合物更具有較佳之粗糙度。透過原子力顯微鏡〈atomic force microscope,AFM〉，利用原子間的相互作用力以得知表面形貌，並測量薄膜100之奈米級表面粗糙度。經過不同表面粗糙度參數包含均方根偏差法〈RMS,Rq〉、算數平均偏差法〈Ra〉以及最大高度法〈Rmax〉計算之輪廓高度參數，薄膜100之表面粗糙度大於100奈米之間。薄膜100之良好粗糙度或者說不平整的表面有利於細胞依附，縮短蒐集時間以及整體檢測時間。 The polymer formed from polyethylene glycol and a polymer having affinity with polyethylene glycol has a better roughness. The atomic force microscope (AFM) was used to understand the surface topography by the interaction force between the atoms, and the nanometer surface roughness of the film 100 was measured. After different surface roughness parameters include the root mean square deviation method <RMS, Rq>, the arithmetic mean deviation method <Ra>, and the maximum height method <Rmax> calculated contour height parameter, the surface roughness of the film 100 is greater than 100 nm. . The good roughness or uneven surface of the film 100 facilitates cell attachment, shortening collection time and overall detection time.
薄膜100經過改質後包含能夠共價、非共價或共價及非共價組合連接之官能基，該等官能基可與連結分子120鍵結或直接連接於生物活性組成130，例如羧基或胺基化後和生物活性組成130結合。根據本發明一實施例，薄膜100表面利用電漿處理，所採用的電漿功率為300W，利用真空系統將腔體的壓力下降到0.001托〈torr〉，再以相同的流量通入氧氣以及氬氣，直到腔體達到穩定壓力0.1托，此時將電源打開產生電漿，持續五分鐘將電源關閉，通入空氣直到1大氣壓，即可將薄膜100取出，完成電漿處理程序。薄膜100經過電漿處理，可保持纖維表面的結構，不至於被破壞，且使得連結分子120以及生物活性組成130可輕易附著於薄膜100。 The film 100 is modified to include functional groups capable of covalent, non-covalent or covalent and non-covalent combination, and the functional groups may be bonded to the linking molecule 120 or directly to the biologically active composition 130, such as a carboxyl group or After amination, it binds to the bioactive composition 130. According to an embodiment of the present invention, the surface of the film 100 is treated by plasma, and the power of the plasma is 300 W. The pressure of the cavity is lowered to 0.001 Torr (torr> by a vacuum system, and oxygen and argon are introduced at the same flow rate. Gas, until the cavity reaches a steady pressure of 0.1 Torr, at this time the power is turned on to generate plasma, the power is turned off for five minutes, and the air is introduced until 1 atmosphere, the film 100 can be taken out, and the plasma processing procedure is completed. The film 100 is plasma treated to maintain the structure of the fiber surface without being destroyed, and the bonding molecules 120 and the bioactive composition 130 can be easily attached to the film 100.
連結分子120接合薄膜100與生物活性組成130且包含官能基，該等官能基能夠共價、非共價或共價及非共價組合連接薄膜100存在之官能基且直接連接於作為生物活性組成之一部份的官能基。連結分子120可以為蛋白質，並且包含特定官能基，例如：羥基、胺基、羧酸、酯基等與薄膜100與生物活性組成130中存在的官能基反應。連結分子120的反應為EDC/NHS反應，此類反應機制使得NH2與COOH官能基產生共價鍵結，根據本發明部分實施例，連結分子120包含NH2官能基，因此在薄膜100上利用電漿產生COOH官能基即可將連結分子120經由EDC/NHS反應固定在表面上。連結分子120對於生物活性組成130有 特殊靜電吸附力，由於氫鍵與靜電吸附力使得生物活性組成130可以透過連結分子120穩定地固定在薄膜100表面。 The linking molecule 120 bonds the film 100 to the bioactive composition 130 and comprises functional groups capable of covalently, non-covalently or covalently and non-covalently linking the functional groups present in the film 100 and directly attached to the bioactive composition. One part of the functional group. The linker molecule 120 can be a protein and comprises a specific functional group, such as a hydroxyl group, an amine group, a carboxylic acid, an ester group, etc., which reacts with the functional groups present in the film 100 and the biologically active composition 130. The reaction of the linking molecule 120 is an EDC/NHS reaction which causes the NH 2 to covalently bond with the COOH functional group. According to some embodiments of the invention, the linking molecule 120 comprises an NH 2 functional group and is therefore utilized on the film 100. The plasma produces a COOH functional group to immobilize the linking molecule 120 to the surface via an EDC/NHS reaction. The linking molecule 120 has a special electrostatic adsorption force for the biologically active composition 130, and the biologically active composition 130 can be stably fixed to the surface of the film 100 through the linking molecule 120 due to hydrogen bonding and electrostatic adsorption.
生物活性組成130接合於連結分子120或薄膜100，且包含用於偵測生物物質或循環腫瘤細胞之結合部分。連結分子120可使用蛋白質而生物活性組成130為抗體，此種抗體對表皮細胞產生專一性的生物免疫反應。生物活性組成130包含官能基，該等官能基能夠共價、非共價或共價及非共價組合連接薄膜100存在之官能基或直接連接於作為連結分子之一部份的官能基。生物活性組成130與生物物質經由分子識別、化學親和力或幾何/形狀識別具有特定親和力。生物活性組成130用於探測生物物質之結合部份包含，例如：合成聚合物、細胞外基質蛋白、結合受體、抗體或對生物物質呈現高親和力之任何其他表面標記。根據本發明一實施例，生物活性組成130結合部分係指anti-EpCAM，其對於循環腫瘤細胞具有高特定性但不存在於血細胞中。 The bioactive composition 130 is bonded to the linking molecule 120 or the membrane 100 and comprises a binding moiety for detecting biological material or circulating tumor cells. The linker molecule 120 can use a protein and the biologically active component 130 is an antibody that produces a specific biological immune response to epidermal cells. The bioactive composition 130 comprises functional groups capable of covalently, non-covalently or covalently and non-covalently linking the functional groups present in the film 100 or directly attached to a functional group as part of the linking molecule. The biologically active composition 130 and the biological material have specific affinities via molecular recognition, chemical affinity or geometry/shape recognition. The bioactive composition 130 is used to detect binding moieties of biological material, for example: synthetic polymers, extracellular matrix proteins, binding receptors, antibodies, or any other surface marker that exhibits high affinity for biological material. According to an embodiment of the invention, the bioactive composition 130 binding moiety refers to anti-EpCAM, which is highly specific to circulating tumor cells but not present in blood cells.
高分子纖維癌症檢測試片10之構件簡單，透過薄膜100之聚乙二醇和與聚乙二醇具有親和性之高分子形成之聚合物達到良好表面粗糙度、不水解、孔隙緊密等特性可以有效滯留細胞，再加上生物活性組成130對於生物物質之特定性，可將特定細胞保留在高分子纖維癌症檢測試片10上，進行進一步檢測判斷。 The polymer fiber cancer detecting test piece 10 has a simple structure, and the polymer formed by the polyethylene glycol of the film 100 and the polymer having affinity with polyethylene glycol can achieve effective surface roughness, non-hydrolysis, and tight pores. The retained cells, together with the specificity of the biologically active composition 130 for the biological substance, can be retained on the high molecular fiber cancer test strip 10 for further detection and judgment.
本發明更提供一種高分子纖維癌症檢測試片作為篩檢特定細胞之用途。該用途包含製備一血液樣本，透過 離心處理之後，取得其中之棕黃層。棕黃層主要包含白血球以及腫瘤循環細胞〈Circulating tumor cells〉。之後，加入適當濃度、對於循環腫瘤細胞有特定性之螢光試劑進行培養。培養程序係指常見實驗室生物細胞培養環境、流程，為本領域具有通常技藝人士所知，因此不在此贅述。再來，將培養之細胞均勻地滴於高分子纖維癌症檢測試片10。待反應結束後可透過螢光顯微鏡作進一步分析。 The present invention further provides a use of a polymer fiber cancer test strip as a screening specific cell. The use comprises preparing a blood sample through After centrifugation, the brownish yellow layer was obtained. The buffy coat mainly contains white blood cells and Circulating tumor cells. Thereafter, the fluorescent reagent having a specific concentration and specificity for circulating tumor cells is added and cultured. The culture program refers to a common laboratory biological cell culture environment and process, and is known to those skilled in the art, and therefore will not be described herein. Then, the cultured cells were uniformly dropped on the polymer fiber cancer test strip 10. After the reaction is completed, it can be further analyzed by a fluorescence microscope.
請參考第6與第7圖，在明暗兩種視野下觀察高分子纖維癌症檢測試片10上之細胞。第6圖為暗視野下散發螢光之區域，第7圖為明視野下相同區域〈圓圈處〉的確為生物物質。因此可確定高分子纖維癌症檢測試片10所捕捉到的細胞為循環腫瘤細胞。由第7圖可見，高分子纖維癌症檢測試片10之表面並無其他細胞干擾視野或遮蔽循環腫瘤細胞，欲觀察之特定生物物質清晰可見。由此可見，利用高分子纖維癌症檢測試片10篩檢特定細胞可有效減少干擾或非特定細胞附著。 Please refer to the 6th and 7th figures to observe the cells on the polymer fiber cancer test strip 10 in both the light and dark fields. Figure 6 shows the area where the fluorescence is emitted in the dark field, and Figure 7 shows the same area in the bright field (the circle) is indeed the biomass. Therefore, it can be confirmed that the cells captured by the polymer fiber cancer detecting test piece 10 are circulating tumor cells. As can be seen from Fig. 7, no other cells interfere with the visual field or mask the circulating tumor cells on the surface of the polymer fiber cancer test strip 10, and the specific biological substance to be observed is clearly visible. Thus, screening of specific cells using the polymeric fiber cancer test strip 10 can effectively reduce interference or non-specific cell attachment.
請參考第5圖。第5圖為高分子纖維癌症檢測試片10臨床試驗總結圖。圖中羅馬數字代表不同大腸癌期數，「I」代表第一期、「II」代表第二期，以此類推。對照組為大腸癌檢測陰性之受試者。受試者血液經過上述方法處理、培養並透過本發明之高分子纖維癌症檢測試片10分析，7件對照組樣本中，循環腫瘤細胞的數量幾乎皆為0或不超過2顆循環腫瘤細胞/毫升。在第二、三以及四期的大腸癌患者樣本中，循環腫瘤細胞數目明顯增加。尤其再第二期 與第三期患者樣本中，高分子纖維癌症檢測試片10甚可捕捉到9顆循環腫瘤細胞/毫升。由以上臨床實驗結果可知，對於在第二、三以及四期的大腸癌的篩檢成功率幾乎為100%。由此可見，高分子纖維癌症檢測試片10在臨床上可成功捕捉血液中極少量的循環腫瘤細胞，且在一天之內即可獲得結果，不僅縮短篩檢時間，也簡化篩檢流程及所需的人力。 Please refer to Figure 5. Figure 5 is a summary of the clinical trial of the polymer fiber cancer test strip 10. The Roman numerals in the figure represent the number of different colorectal cancers. "I" stands for the first period, "II" stands for the second period, and so on. The control group was a subject with a negative colorectal cancer test. The blood of the subject was treated, cultured and analyzed by the above-mentioned polymer fiber cancer test strip 10 of the present invention. In the 7 control samples, the number of circulating tumor cells was almost 0 or no more than 2 circulating tumor cells/ ML. In the second, third and fourth stages of colorectal cancer patients, the number of circulating tumor cells increased significantly. Especially the second period In the third-phase patient sample, the polymer fiber cancer test strip 10 could capture 9 circulating tumor cells/ml. From the results of the above clinical experiments, it is known that the screening success rate for colorectal cancer in the second, third and fourth phases is almost 100%. It can be seen that the polymer fiber cancer test strip 10 can successfully capture a very small amount of circulating tumor cells in the blood in a clinical manner, and the results can be obtained within one day, which not only shortens the screening time but also simplifies the screening process and the laboratory. The manpower required.
雖然本發明已以實施方式揭露如上，然其並非用以限定本發明，任何熟習此技藝者，在不脫離本發明之精神和範圍內，當可作各種之更動與潤飾，因此本發明之保護範圍當視後附之申請專利範圍所界定者為準。 Although the present invention has been disclosed in the above embodiments, it is not intended to limit the present invention, and the present invention can be modified and modified without departing from the spirit and scope of the present invention. The scope is subject to the definition of the scope of the patent application attached.
10‧‧‧試片 10‧‧‧ test strips
100‧‧‧薄膜 100‧‧‧film
120‧‧‧連結分子 120‧‧‧Linking molecules
130‧‧‧生物組成 130‧‧‧ Biological composition
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| Pant HR et al., "Formation of electrospun nylon-6/methoxy poly(ethylene glycol) oligomer spider-wave nanofibers", Materials Letters, vol.64, p.2087-2090, 2010/07/01 * |
| Yoon HJ et al., "Nanoassembly of graphene oxide for circulating tumor cell isolation", 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences October 2-6, 2011, Seattle, Washington, USA * |
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