Fabrication of Microfilters and Nanofilters and Their Applications

Abstract

Micro- and nanofilters with precision pore sizes and pore layout have applications in many fields including capturing circulating tumor cells and fetal cells in blood, water treatment, pathogen detection in water, etc. Methods to fabricate micro- and nanofilters not using track etching or reactive ion etching are provided, allowing easy fabrication of single layer or stack of films simultaneously, and/or stack of films on rolls. Microfilter can be made using one or more layers of material. Invention enables mass production of microfilters with lithographic quality at low cost. Isolation, enumeration and characterization of circulating tumor cells using microfilters provides (i) guides to cancer treatment selection and personalize dosage, (ii) low cost monitoring for treatment response, disease progression and recurrence, (iii) assessment of pharmacodynamic effects, (iv) information on mechanisms of resistance to therapy, and (v) cancer staging. Microfabrication methods are also applicable to fabrication of any free standing patterned polymeric films.

1 . A microfabrication method comprising:
 patterning and etching predetermined pore sizes, distributions and shapes in one or more layers of polymer films simultaneously using ultra-violet lithography or x-rays lithography;   optionally stacking one or more layers of the film on an individual wafer; and   exposing one wafer at a time.   
     
     
         2 . The method of  claim 1  further comprising automation, wherein:
 one or more layers of the films are supplied in rolls; 
 for each exposure, the films advance by the appropriate amount; 
 during the exposure, the films are held securely in place; 
 after the exposure, the film advances and process repeats; and 
 after exposure, the exposed films are developed to form the pores. 
 
     
     
         3 . The methods as claimed in  claim 1  wherein the film comprises any free standing patterned polymeric films. 
     
     
         4 . A method of utilizing microfilters as claimed in  claim 1  made from one or more of a variety of polymer materials, other than solid parylene, applied to enrich CTCs from peripheral blood in cancer patients 
     
     
         5 . A method of utilizing microfilters as claimed in  claim 1  made from one or more of a variety of polymer materials, applied to enrich primitive fetal nucleated red blood cells in peripheral blood of pregnant women, 
     
     
         6 . A method of utilizing precision microfilters as claimed in  claim 1  comprising any one of: water filtration; beer and wine filtration; pathogen detection; capture of circulating tumor cells in peripheral blood of cancer patients and primitive fetal nucleated red blood cells in peripheral blood of pregnant women; enriching stromal cells, mesenchymal cells, endothelial cells, epithelial cells, stem cells, and non-hematopoietic cells from blood samples and tumor or pathogenic cells in urine. 
     
     
         7 . A method of utilizing microfilters as claimed in  claim 1  comprising: isolating and detecting rare cells from a biological sample containing other matter and other types of cells. 
     
     
         8 . The method of  claim 7  further comprising: filtering fluid samples using the microfabricated filters; and performing downstream processes using the enriched cells, the processes including identification, characterization, or growth in culture. 
     
     
         9 . The method of  claim 7  further comprising subjecting the final enriched target cells to a variety of analyses and manipulations including staining, immunofluorescence, cell counting, PCR, fluorescence in-situ hybridization (FISH), immunohistochemistry, flow cytometry, immunocytochemistry, image analysis, enzymatic assays, gene expression profiling analysis, efficacy tests of therapeutics, culturing of enriched cells, and therapeutic use of enriched rare cells. 
     
     
         10 . The method of  claim 9  further comprising, optionally recovering and subjecting the depleted plasma protein and white blood cells to other analysis including inflammation studies and gene expression profiling. 
     
     
         11 . A method of utilizing microfilters as claimed in  claim 1  comprising isolating and detecting rare cells from a biological sample containing other types of cells. 
     
     
         12 . A method of utilizing microfilters as claimed in  claim 1  comprising: capturing circulating fetal cells in the mother's blood during 11-12 weeks of pregnancy, wherein the fetal cells circulating in the peripheral blood of pregnant women are a potential target for noninvasive genetic analyses, and the cells include epithelial (trophoblastic) cells, which are 14-60 μm in diameter, larger than peripheral blood leukocytes. 
     
     
         13 . A method of utilizing microfilters as claimed in  claim 1  comprising enrichment circulating fetal cells followed by genetic diagnostic can be used for noninvasive prenatal diagnosis of genetic disorders using PCR analysis of a DNA target or fluorescence in situ hybridization (FISH) analysis of genes. 
     
     
         14 . A method of utilizing microfilters as claimed in  claim 1  comprising: detecting of circulating tumor cells in blood, wherein the microfilters are fabricated with precision ordered pores using a large number of available polymer materials, the materials comprising a thin coating of parylene. 
     
     
         15 . A method of utilizing microfilters as claimed in  claim 1  comprising: obtaining a blood sample from the patient, which can be in the range of 5-10 ml; and flowing the blood through the microfilter; wherein the microfilter is held in a filter holder with an inlet, an outlet, by securely holds the filter around the edges. 
     
     
         16 . A device comprising the microfilter as claims in  claim 1 , further comprising: a filter holder; and build in support in the filter holder, wherein the holder optionally comprises a gasket. 
     
     
         17 . A method of utilizing microfilters as claimed in  claim 15  further comprising: pushing the blood through from the inlet, wherein: most cells larger than the pore dimension are retained; some white blood cells are deformable and can pass through pores with smaller dimension than the cell size; and the application of enriching circulating fetal cells and tumor cells are based on the principle associated with the retaining and passing through of the cells. 
     
     
         18 . A method of utilizing microfilters as claimed in  claim 1  wherein the microfilter pores can be larger than 7-8 μm in diameter for enriching CTCs for cancer with large cancer cells and can be smaller for cancer with smaller cancer cells 
     
     
         19 . A method of fabricating microfilters as claimed in  claim 1  wherein the microfilter are 10-25 μm, and thicker microfilters provide more structural strength. 
     
     
         20 . A method of utilizing microfilters as claimed in  claim 1  wherein the captured CTCs can be enumerated on the microfilter and can be specifically identified by genomic DNA and fluorescently tagged antibodies for intracellular and surface markers. 
     
     
         21 - 49 . (canceled)