conventional methods of generating porous si nanostructures that may then be used in separating and detecting analytes such as macromolecules , proteins , protein fragments , polymers , biopolymers , biomolecules , small molecules , biological cells or other molecular complexes , include inherent drawbacks , notably that conventional methods are slow and require several sequential steps in order to detect and separate a particular analyte . use of porous silicon ( si ) to separate and detect these analytes provides an advantageous alternative to conventional methods in that the porosity and pore size of si may be readily tuned and configured to suit a particular application , and could be used in varying applications spanning biohazard and bioweapons detection , to in vivo detection of viral proteins . in addition , porous si may be used the separate and detect analytes embodiments of the invention . furthermore , porous si has the added advantage of being conducive to being configured in a compact format , such as a chip , a wafer , or other types of thin film , making porous si amenable to applications placing a premium on small size and transportability . additionally , using si as a matrix for detection and separation provides a faster method in that simultaneous detection and separation is possible , and its use is conducive to integration with other analytical tools , such as matrix assisted laser desorption ionization , or maldi . in further embodiments of the invention , porous si is be configured to allow admission and controlled release of a relatively small drug . nanocrystalline porous si films may be employed both as biosensors ( for unlabeled proteins and deoxyribonucleic acid , or dna ) and as chromatography matrices , and as such , are useful in coupling the detection and separation processes as simultaneous events . the instant invention includes use of porous si for the simultaneous detection and separation of analytes , including macromolecules , proteins , biomolecules and other molecular complexes , using a porous si layer that has been electrochemically machined to present a range of pore sizes to an analyte solution . while numerous analytes may be separated and detected using embodiments of the instant invention , for exemplary purposes , a test molecule , bovine serum albumin ( bsa ), will be discussed herein . the admittance of bsa in the porous si film is preferably monitored using the optical interference technique to demonstrate the existence of a critical pore size , above which a characteristic change in the optical index of the film is detected . in addition , a critical relationship between surface charge and isolectric point ( pi , ph at which the molecule has no net charge ) of the protein is monitored for purposes of separation and detection . porous si films displaying a distribution of pore dimensions may preferably be generated by electrochemically etching si in aqueous ethanolic hf using an asymmetric electrode configuration . the porous si film may be nanomachined to have a predetermined configuration in that the median pore size and breadth of the size - distribution in the si film may optionally be set by adjusting the hf concentration , current density , and position of the counter electrode relative to the si electrode . preferably , si films with pore sizes in the range of a few nanometers are used as size - exclusion matrices , and are capable of performing an “ on - chip ” determination of macromolecule dimensions . advantageously , embodiments of the instant invention allow formation of different pore sizes across an si film during a single process . pore size variation may be configured specifically to a target analyte . for example pores having a diameter ranging from between 2 and 10 nanometers may be configured to allow admission and controlled release of a relatively small drug . pores having a diameter ranging from 10 to 50 nanometers may be configured for separation of protein molecules , pores having a diameters ranging from 50 to 300 nanometers may be configured for separation of viral particles , and pores having diameters ranging from 500 nanometers to 5 microns may be configured to admit and separate bacterial cells . more specifically , the chip or other si film substrate may be exposed to an environment suspect of including a particular analyte , especially a protein , such as by being immersed in a suspect solution or by exposing the chip to air or vapors above a reactor . exposure may include immersing the chip in the vapor , or simply flowing the vapor over the chip . analysis of the exposed chip will reveal the size of the of the target analyte , by an examination of the reflectivity spectra . as an example , using the test molecule , bsa , optical reflectivity spectra of the thin porous si films display distinctive shifts in the fabry - perot fringes in regions of the si film where the pore dimensions are larger than a critical size , which is interpreted to be the characteristic dimensions of the protein . gating of the protein in and out of the porous films is preferably achieved by adjustment of the solution ph below and above the pi ( isoelectric point ) value , respectively , though it can also be achieved by adjustment of a bias potential on the si sample , or alternatively by changing the temperature of the sample . further discrimination can also be achieved by addition of selective binding agents , such as antibodies , for the analyte . porous si is a nanocrystalline matrix that may be formed on single crystal si film , such as a chip , wafer , or other thin film in an electrochemical etch . formation of porous si involves the electrochemical oxidation of si in a solution containing hydrofluoric acid ( hf ). the properties of the porous layer , such as pore size and porosity , are dependent on a number of factors , such as hf concentration , dopant type and density in the si substrate , and current density used in the electrochemical etch . exemplary dopants include boron , antimony , or phosphorus , with exemplary concentrations varying from between approximately 10 − 22 cm − 3 and 10 − 16 cm − 3 . similarly , exemplary current density may range from 1 microamp per cm 2 and 2 amp per cm 2 . a particular advantage of a formation method according to an exemplary embodiment is the ability to form reproducible porous si micro - and nano - architectures , which has contributed to successful incorporation of these structures in biosensors and optoelectronic components . while methods of detection may vary to suit individual applications , a preferred detection modality is measurement of the fabry - perot fringes that result from optical interference between the top and bottom surfaces of the porous si film . thus , by monitoring changes in the refractive index of the si film , volume and molecular weight of a target analyte may be readily identified . preparation and characterization of porous si films possessing lateral pore gradients for purposes of the preferred embodiment , porous si films containing a distribution or pore sizes may be electrochemically prepared using the asymmetric electrode configuration illustrated in fig1 . turning therefore to fig1 for exemplary purposes , a diameter of an o - ring is illustrated to be approximately 1 . 3 cm . a counter electrode is preferably placed ˜ 1 mm from the si working electrode surface and ˜ 1 mm from the edge of the o - ring . the value of x used in fig1 refers to a distance from a point on the o - ring edge closest to a pt counter electrode to a position on the si surface as illustrated , and the pt counter electrode is disposed at approximately x = 1 mm . in this arrangement , a potential at the si electrode - solution interface varies as a function of distance from the pt counter electrode due to solution resistance , leading to a decrease in current density as the distance from the counter electrode increases . pores ranging in size from 2 - 1500 nm may be obtained in highly boron - doped si substrates ( p **) by varying such factors as the current density , and that pore size may also determined by the ratio of aqueous hf / ethanol used . the asymmetric electrode configuration provides a current density gradient across the wafer that results in a distinct and reproducible variation in the size of the pores . this method is particularly advantageous in that it quickly prepares a material that has a predetermined range of pore sizes . conventional methods for preparing materials containing a range of pore or void sizes are cumbersome , unduly expensive and relatively slow . in contrast , embodiments of the instant invention provide for preparation of a porous sample having a distribution of pore sizes in the same monolithic substrate simultaneously , whereas in previous methods separate steps would need to be undertaken to generate the same distribution of pore sizes . in addition , a center and breadth of this pore size distribution can be controlled . this has been demonstrated experimentally . more specifically , to demonstrate the instant invention , three circular film samples of porous si may be prepared in separate experiments wherein an hf ( aq )/ ethanol ratio in the electrolyte used to prepare these samples is varied between 1 : 1 , 1 : 2 , and 1 : 3 , respectively and each circular sample is configured to be approximately 1 . 2 cm in diameter . by applying a current density of 115 ma / cm2 for 8 seconds to each of the three crystalline silicon samples , each of the resulting porous samples contains a distribution of pore sizes . tapping - mode atomic force images reveal an approximately linear distribution of pore sizes in all three samples , and the maximum pore size , minimum pore size , and center of the size distribution is different for all three samples for a given electrode geometry , the properties of the si pore gradient film may further be varied by varying the electrolyte composition . for example , almost no electropolished region is observed on the si sample that was etched in hf ( aq )/ etoh = 3 : 1 solution , and the range of pore sizes ( as determined from atomic force microscopy , or afm , measurements , not included ) is less pronounced than in samples etched in hf ( aq )/ etoh = 1 : 1 and 2 : 1 , respectively . in hf ( aq )/ etoh = 2 : 1 solutions , a film with a wider range of pore sizes is formed . some of the porous film directly beneath the electrode position generally peels upon drying , but afm measurements on the intact film reveals pore diameters ranging from ˜ 600 nm adjacent to the peeled spot to & lt ; 10 - 20 nm ( afm probe tip limited features ) about 7 mm from the original counter electrode position . profilometry measurements reveal that the thickness of the porous si layer varies as a function of distance from the counter electrode , as illustrated in fig3 . the counter electrode was directly above the si film at x = 1 . 5 mm . film thickness was measured by profilometry of the si substrates after removal of the porous layer . in the exemplary embodiment , the etch conditions were as follows : 115 ma / cm 2 , 90 seconds , hf ( aq )/ etoh = 1 . 5 : 1 . to obtain the film thickness data the porous si was removed by treatment with a basic solution ( koh / h 2 o / isopropanol = 5 : 16 : 4 by volume ) for about 10 seconds . a similar depth profile was observed for all of the etch conditions employed and varied only moderately as a function of electrolyte composition . the films are generally thickest at the point directly under the counter electrode , consistent with that being the position of highest current density . the si dissolution rate and hence the rate of film growth is directly proportional to the current density . thus , the profilometry data provide an indirect measure of the current density profile . the current density profile for the etch an also be estimated by applying ohm &# 39 ; s law to the electrode configuration and using reasonable values for the solution resistance , as illustrated in fig2 . for the electrochemical cell used in this embodiment , the current density varies from a value of ˜ 200 ma / cm 2 directly under the electrode to less than ˜ 50 ma / cm 2 at the far edge . thus the porosity and depth variations observed in fig2 are consistent with a current density variation that results from ohmic ( ir , current - resistance voltage ) drop in the solution . in regions of the film where the pore dimensions are too small to effectively scatter light , optical interference occurs between the air / porous si and the porous si / bulk si . the reflection spectra of these films are described by the fabry - perot relationship and appear as a series of reflectivity maxima , as illustrated in fig3 . a wavelength of maximum constructive interference for the mth order fringe in a film of thickness l and refractive index n is given by the following equation : the quantity 2nl is herein referred to as the effective optical thickness ( eot ). a change in the refractive index ( n ) that occurs upon introduction of molecular adsorbates , which are preferably the analyte for the particular application , in the porous si film results in a shift in the optical fringes , which has been measured with a high sensitivity using either a spectrometer with a white light source , or a photodiode with a laser source . spatially resolved spectra were obtained using a microscope objective as a collection optic with a spatial resolution of approximately 0 . 3 mm 2 . the spectra ( taken in air ) were obtained from spots on the si wafer at approximately 2 mm ( a ), 5 mm ( b ), and 9 mm ( c ) from the counter electrode during preparation . etch conditions were 115 ma / cm 2 for 90 seconds , 3 : 1 hf ( aq )/ etoh electrolyte . fig3 illustrates representative spectra from three points on a chip . as illustrated in fig3 , the spacing of the fabry - perot spectra obtained from samples that contain pore gradients vary across the length of the porous film because of variations in both depth and porosity . the variation in fidelity is ascribed to changes in refractive index contrast at the porous si / air and porous si / crystalline si interfaces . the corresponding change in eot is obtained from the fourier transform of the intensity vs . frequency plot of the reflectivity spectrum ( applying a hanning window function to eliminate edge artifacts .). impregnation of biomolecules into a porous si fabry - perot layer results in an increase in eot because a certain volume of the thin film once occupied by the aqueous buffer solution is now occupied by the biomolecule , which has a larger index of refraction ( e . g ., n buffer = 1 . 33 , n protein = 1 . 42 ). a film containing a range of pore diameters is expected to exhibit a change in eot upon exposure to an analyte only in the regions of the film containing pores that are large enough to admit the analyte . in the present work , bsa was used as a probe analyte . bsa has an average molecular weight of 68 kd with its isolectric point at ph 5 . the data illustrated in fig4 a and 4b represent the difference between the value of eot measured in a given eluent and the eot value measured in air , as a function of position ( x ) along the porous si gradient . in both fig4 a and 4b , the largest pores in the gradient are at position 0 mm , and the smallest are at position 10 mm . each measurement was made as a function of position on the pore gradient wafer , and is relative to the edge of the wafer that was closest to the pt counter electrode used in the etch . fig4 a specifically illustrates eot measurement in the presence of ph 4 buffer ( o ), bsa in ph 4 buffer ( ), and ph 7 . 2 pbs buffer (⋄), relative to the measurement in air at the corresponding position . fig4 a specifically illustrates the difference between the two curves representing bsa in ph 4 solution ( in fig4 a ) and pure buffer ( ph 4 ) solution ( o in fig4 a ). the data indicate that bsa is admitted into the pores residing at positions between 0 and 4 mm from the film edge ; at positions between 4 and 10 mm the pores are too small and bsa is excluded . lines are included as a guide to the eye . the positive change in eot ( δeot ) values for all three solutions ( ph 4 buffer , bsa in ph 4 buffer , and ph 7 . 2 phosphate buffered saline , pbs , buffer ) demonstrate that the film is porous throughout its length . the change in eot tends to be largest near x = 0 ( edge closest to the counter electrode position ) for all solutions because the film is thickest and has the highest porosity at this point , resulting in the largest changes in eot relative to air . fig4 b illustrates the difference observed in the chip with and without added bsa , obtained by subtracting the bsa ( in ph 4 buffer ) curve from the ph 4 buffer curve of fig4 a . introduction of the bsa results in an increase in the relative values of eot in the region of the chip between 0 and 4 mm . thus , the region of the porous si film at 0 & lt ; x & lt ; 4 mm is interpreted to be the region in the gradient in which the pore size is large enough to allow entry of the bsa molecule . the response tapers off in the region 1 & lt ; x & lt ; 4 mm , and no relative change is seen for bsa at x & gt ; 5 mm , where the pores are presumably too small to admit bsa into the film . oxidation converts the porous si hydride - terminated surface into an oxide that is negatively charged even at moderately low ph values . in contrast , the charge on the bsa molecule can be changed by appropriate adjustment of the ph . thus the electrostatic interaction between the sio 2 - covered pore surface and the protein can be varied , allowing the separation of molecules based on their pk a values . the isoelectric point of bsa is approximately 5 . thus , at solution ph values above 5 , the protein is negatively charged and below ph 5 the molecule is positively charged , due to pk a values of the constituent amino acid residues . in the size - exclusion experiments illustrated in fig5 , the ph of the system was maintained at 4 , where the oxidized porous si sample is expected to have a net negative charge and bsa is expected to have a net positive charge . however , if the ph is raised such that the protein becomes negatively charged , the biomolecule can be excluded from the porous silicon matrix . the result of adjusting the solution ph is demonstrated in fig5 . the sample used in this embodiment had pores that were large enough to accommodate bsa protein at ph 4 . at time = 0 ( a in fig6 ), aqueous pbs buffer ( ph 7 . 2 ) is introduced into the flow cell . at point b , a solution of bsa in pbs buffer is added to the eluent stream . no change is observed in the eot value , indicating that at this ph no protein is admitted to the pores . the flow cell is flushed with pure pbs buffer solution at ph 7 . 2 to remove residual bsa ( c ), and the buffer solution is then changed to potassium biphthalate buffer at ph 4 ( d ). once the ph of the solution is lowered to the point where bsa has a net positive charge , bsa is introduced ( e ) and the value of eot is observed to increase , corresponding to the admission of the protein into the porous silicon matrix . at point f , the solution is changed back to pure potassium biphthalate buffer ( ph 4 ) containing no bsa . after this point the value of eot is observed to gradually decrease , indicating that the protein trapped in the porous silicon matrix is slowly diffusing out . at point g , the pbs buffer solution is introduced , resulting in a rapid drop in the eot value as the remaining bsa diffuses out of the pores . desorption of bsa is strongly promoted in the ph 7 . 2 pbs buffer solution , presumably because the negatively charged protein is repelled by the negatively charged si oxide surface . the reproducibility of the process may be probed in a second run , where bsa in ph 4 buffer is reintroduced ( h ) and subsequently removed with a pbs buffer rinse ( i ). significant improvements in surface stability have been achieved by appropriate chemical modification of the porous si surface , such as thermal hydrosilylation , photochemical hydrosilylation , or electrochemical grafting of species to the porous si surface by means of si — c bonded linkages . notably , no bsa adsorption is observed when bsa is introduced in a ph 7 . 2 pbs buffer solution ( fig5 , point b ), and that no significant change in the eot value is observed when the ph of the solution is changed from 4 to 7 . 2 in the absence of the protein ( fig4 a , 4b and 5d ). porous si films can be prepared that contain a controlled distribution of pore sizes using anisotropic etching conditions . the inhomogeneous etch results in a porous si film having pore diameters that vary laterally and span a range of sizes from & lt ; 10 nm to & gt ; 500 nm . as illustrated in the drawings , the simultaneous separation and detection of a protein in a nanomachined si matrix is achieved , as is gating of the response by adjustment of ph below and above the isoelectric point of the protein . this provides an additional means of analyte separation and identification . thus , the instant invention simultaneously detects and separates the target analyte , which may then be subjected to further analysis . embodiments of the instant invention are especially advantageous for determination of protein size and for the detection of weakly - bound complexes . additionally , the ability to controllably trap and release analytes such as proteins may be useful for drug delivery applications , as porous si has been shown to be biocompatible and readily bio - resorbable . more specifically , the porous si film may be configured to have a predetermined porosity and predetermined pore size such that a specific drug having a specific molecular weight and a specific volume takes a predictably specific time to enter into and subsequently be released from a pore . in this manner , the specific drug may be time released in vivo . more specifically , the porous si film may be configured to have a range of pore sizes that promote a predetermined release profile , defined as the moles of drug released from the porous si film versus time . the particular drug used in the instant embodiment may include anticancer drugs , chronic pain medication , anti - inflammatory drug such as dexamethasone , an anti - coagulant , an anti - rejection drug such as rapamycin ( which is used in coronary artery stents to inhibit restenosis ). the porous si film , and therefore the drug , may be introduced through a variety of mechanisms , including transdermally , via intramuscular injection , intravenously , surgically implanted , through the throat or lungs , or orally ingested . another particular application of the instant invention involves proteomics where a solution may contain a complex mixture of proteins , but wherein it is desirable to isolate or purify a single protein within the mixture . for example , of particular interest are proteins that are produced by an undesirable genetic mutation , proteins that act as markers , or indicators , of a specific disease state , or proteins which currently have no known function but may be key in the future diagnosis or treatment of a disease . once the mixture is subjected to the detection and separation processes of the instant invention , the target protein could then subsequently be subjected to processing in a maldi instrument to refine identification of the molecule on the basis of high resolution mass spectrometry . while various embodiments of the present invention have been shown and described , it should be understood that other modifications , substitutions and alternatives are apparent to one of ordinary skill in the art . such modifications , substitutions and alternatives can be made without departing from the spirit and scope of the invention , which should be determined from the appended claims . various features of the invention are set forth in the following claims .
a method of controlled in vivo drug delivery is provided . a porous silicon matrix having pores sized and configured to admit to trap and then release a predetermined molecular complex with a predetermined dose - time profile is selected . the matrix contains the predetermined molecular complex so that the predetermined molecular complex is disposed within the pores of the porous silicon matrix . the matrix is introduced into a human body . the drug releases according the dose - time profile . the introduction can be via transdermal introduction , intramuscular injection , intravenous introduction , surgical implantation , inhalation , and oral ingestion .