WO2010048627A2 - Potentiometric sensors based on surface molecular imprinting, and nanoparticle composition for the detection of cancer biomarkers and viruses and methods of use thereof - Google Patents

Abstract

Disclosed is a method and apparatus using surface molecular imprinting and nanoparticles for the rapid determination of cancer biomarkers, detection of viruses, and the treatment of cancer, via a treatment that selectively targets cancer cells.

Description

POTENTIOMETRIC SENSORS BASED ON SURFACE MOLECULAR IMPRINTING,

AND NANOPARTICLE COMPOSITION FOR THE DETECTION OF CANCER

BIOMARKERS AND VIRUSES AND METHODS OF USE THEREOF

PRIORITY This application claims priority to U.S. Provisional Application No. 61/108,061 , filed

October 24, 2008, to U.S. Provisional Application No. 61/108,067, filed October 24, 2009, and to U.S. Provisional Application No. 61/1 19,568, filed December 3, 2008, the contents of each of which is incorporated herein by reference.

1. FIELD OF THE INVENTION The present invention relates generally to the use of novel sensors based on surface

Molecular Imprinting (MI) and nanoparticles for the rapid determination of cancer biomarkers and detection of viruses, for the treatment of cancer, and, more particularly, to treatment involving selective targeting of cancer cells.

2. BACKGROUND OF THE INVENTION Cancer biomarkers are critical not only for the diagnosis of cancer but also for monitoring its response to treatment or recurrence and, more recently, for the assessment of cancer risk. Most cancer biomarkers are either single proteins or protein-based molecular assemblies. Modern high throughput methods have accelerated the pace of biomarker discovery necessitating the development of rapid, simple and reliable assays for their detection and quantification in biological fluids and tissues. Current analytical methods are in general cumbersome or time- consuming; they usually assay a single protein; are often costly; and, in the case of antibody- based assays, there are occasional concerns stemming from the cross-reactivity of the detecting antibody.

It is also well known that a Folate Receptor (FR) is frequently ovcrcxpressed in cancer cells. In particular, three FR isoforms have been identified in human tissues and tumors: FR-a, FR-B and FR- y. FR-a and FR-B are known to be greatly overexpressed in many human tumors, while normal tissues typically express an insignificant level of FR-a and a low level of FR-B. FR-y is typically found only in hematopoietic cells. Consequently, folate has been employed in the selective targeting of cancer cells. Some folate-assisted targeting methods that have been explored include delivery of liposomes, macromolecular anticancer therapeutic agents, carboplatin analogs, radio labeled pharmaceuticals, and gene transfer. However, there remains a need in the art for a folate receptor targeting composition which can readily induce the apoptosis of cancer cells when selectively binding thereto, while also being easier and less costly to synthesize. There is also an increasing need for precise molecular detection as a diagnostic tool for early identification of diseases, pathogens, and abnormal protein levels in the body. Proteins are biologically critical for detection as protein-protein interactions govern much of cell regulation and control. Likewise, detection of biomacromolecules and bacteria must be timely while guaranteeing accuracy. An ideal biosensor is one that has both specificity and sensitivity. One common technique for constructing such biosensors is through the process of MI, in which outlines of a specific molecule, such as a protein, can serve as templates for future detection of the molecule in solution. Matching a host surface to a guest molecule depends on geometric organization of functional groups and domains for each. Likewise, binding of host and guest occurs only if the forces involved in the complex formation yield a decrease in the free energy of the system. Current methods for molecular detection make use of monoclonal antibodies for an Enzyme-Linked Immunosorbent Assay (ELISA). However, this process is expensive, time consuming, fragile, and consists of a labile recognition element. With potential applications in medical devices, biomarker sensing, and drug delivery, Ml is robust and cost-effective in comparison to existing detection techniques.

The continuing discovery of cancer biomarkers necessitates improved methods for their detection. MI using artificial materials provides an alternative to the detection of a wide range of substances. Traditional MI methods for biosensing application are three dimensional, making use of bulk polymer matrices to trap template molecules for imprinting. This technique is unfavorable because of its long response time and low molecular reoccupation of polymer cavities, as the dense polymer matrix impedes the movement of large macromolecules like proteins. Alternatively, MI on two-dimensional surfaces has been proposed through Self- Assembled Monolayers (SAM) of a surfactant alkanethiol (1 1-mercapto-l-undecanol) and gold, which are flexible in design and allow for increasing molecular complexity. See WO 2005/003716 A2 to Levon, et al., the contents of which is incorporated by referenced herein. Though recognizing only one surface of the template molecule, two-dimensional MI is advantageous because of its high mass transfer, simple construction and integration of surface imprinting with a transducer.

Of the various embodiments of MI, Surface-MI (SMI) using SAM is particularly suitable for analytical purposes. However, its application has been limited to the detection of small molecules. To detect big molecules, more complicated two-dimensional imprinting procedures are required. The present invention overcomes the shortcomings of conventional systems by templating with hydroxyl functionalized alkanethiol molecules assembled on a gold-coated silicon chip. The directional polar bond of the template with the hydroxyl groups provides further selectivity and only a small portion of the 'key' needs to be recognized by the 'lock'. The invention provides a sensing element that can also recognize large molecules, such as proteins, at concentrations as low as several ng/mL. SUMMARY OF THE INVENTION

The present invention relates generally to the use of novel sensors based on SMI and nanoparticles for rapid determination of cancer biomarkers and detection of viruses, and for the treatment of cancer, and more particularly, wherein the treatment involves selective targeting of cancer cells. Surface molecular imprinting using self- assembled monolayers is applied to design sensing elements for the detection and quantification of cancer biomarkers and other proteins; in the present invention consisting of a gold-coated silicon chip onto which alkanethiol molecules with hydroxyl end groups are chemically bound through a sulfur-metal bond. Binding of the biomarker to the sensing element provides a varied potential, which can be measured potentiometrically, preferably to detect CarcinoEmbryonic Antigen (CEA) in both solutions of purified CEA and in the culture medium of a CEA-producing human colon cancer cell line.

In one aspect the invention relates to the application of the principles of molecular imprinting to the development of a new method for the detection of protein cancer biomarkers and to protein-based macromolecular structures, such as a virion. In another aspect, the invention is directed to a cancer-treatment composition containing platinum nanoparticle cores coated with Folic Acid (FA) molecules. The composition is herein also referred to as 'the complex'. Preferably, the platinum nanoparticle cores have an average size, i.e. diameter, of no more than about 10 nm.

In another aspect, the invention is directed to a method for the treatment of cancer or a pre-cancerous condition, i.e. targeting of neoplastic cells, by administering an effective amount of the above composition to a mammal afflicted with cancer or a pre-cancerous condition. The effective amount is an amount that preferentially, i.e. selectively, induces cell apoptosis in cancerous or precancerous cells over normal cells. Cancerous and precancerous cells are at times referred to herein as neoplastic cells. Preferably, the ratio of cellular uptake of folate- coated platinum nanoparticles in cancerous cells compared to normal cells is at least approximately a ratio of 2.5 to one. Preferably, the effective amount of folate-coated platinum nanoparticles is an amount that corresponds to an IC₃0 fold value of at least about 1.0.

The invention advantageously provides a folate receptor targeting composition that is facile and inexpensive to synthesize, while being highly effective in selectively binding to and preferentially inducing the apoptosis of neoplastic cells.

BRIEF DESCRIPTION OF THE FIGURES

The above and other objects, features and advantages of certain exemplary embodiments of the present invention will be more apparent from the following detailed description taken in conjunction with the accompanying drawings, in which: Fig. 1 shows thiol and molecule for templating, incubated with a gold chip to yield SAM;

Fig. 2 is a diagram of interconnectedness of cell cytoskeleton and an extracellular matrix; Fig. 4 is an electrochemical potentiometer setup for testing when reintroducing protein to an imprinted chip;

Fig. 5 provides scanning electron microscope images within the thiol layer;

Fig. 6 shows potentiometric response with increasing fibrinogen concentration; Fig. 7 shows selective potentiometric response of fibrinogen-imprinted chips to cross testing with hemoglobin;

Fig. 8 is a graph showing selective potentiometric response of hemoglobin-imprinted chips to cross testing with fibrinogen;

Figs. 9 is a graph showing selective potentiometric response of P aeruginosa-impήnted chips to cross testing with S. aureus;

Fig. 10 is a graph showing selective potentiometric response of 5. ai/ra/s-imprinted chips to cross testing with P. aeruginosa;

Fig. 1 1 is a graph showing potentiometric response of P. aeruginosa- imprinted, with only P. aeruginosa being most responsive; Fig. 12 is a graph showing potentiometric response to fibronectin detection in dermal fibroblasts with and with exposure to Au nanoparticles;

Fig. 13 is a graph showing potentiometric response to fibrinogen detection in dermal fibroblasts with and with exposure to Au nanoparticles;

Figs. 14(a)-(c) show potentiometric responses to osteocalcin detection in Dental Pulp Stem Cells (DPSC) after exposure to varying surfaces, days 3, 5 and 7, respectively;

Fig. 15 is a transmission electron microscope image of folate-coated platinum nanoparticles;

Fig. 16 is a bar chart showing distribution of sizes of the platinum nanoparticles;

Fig. 17 is a graph of a showing results of a tetrazolium salt study on breast cancer cells (MCF7 cells) treated with folate-coated platinum nanoparticles;

Fig. 18 is a graph showing results of the tetrazolium salt study on normal breast cells (MCFlOA cells) treated with folate-coated platinum nanoparticles;

Fig. 19 is a bar chart comparing apoptosis results of MCF7 and MCFlOA cells treated with folate-coated platinum nanoparticles; Fig. 20 is a graph of a Reactive Oxygen Species (ROS) study of MCF7 cells by a

SpectraMax method;

Fig. 21 is a graph of a ROS study of MCFlOA cells by the SpectraMax method; and

Fig. 22 is a graph of a ROS study of MCF7 and MCFlOA cells by flow cytometry.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The following detailed description of preferred embodiments of the invention will be made in reference to the accompanying drawings. In describing the invention, explanation about related functions or constructions known in the art are omitted for the sake of clearness in understanding the concept of the invention, to avoid obscuring the invention with unnecessary detail.

As shown in Fig. 1, a SAM is comprised of a Si wafer, a 50 nm gold layer formed through chemical vapor deposition, a densely-packed crystalline layer of an active thiol surfactant that is approximately 1-3 nm thick and the molecule desired for templating embedded in the thiol. Alkanethiols have strong adsorption onto metal surfaces like gold and silver, and voltammetric tests indicate that the thiol is deprotonated upon adsorption to the gold by the Equation (1 ), described by Wink, T, et al.. Self-assembled Monolayers for Biosensors, The Analyst, 122: 43R-50R (1997). R-S-H + Au < > R-S-Au + e + H+ ( 1 )

When the gold chip is incubated with thiol and the molecule intending for imprinting, this SAM is usable to sense proteins and bacteria, as well as for in vitro detection of cell processes and functionality. Gold-thiol assemblies imprinted with desired molecules were additionally examined under the atomic force microscope and scanning electron microscope in order to better understand the surface mechanics behind MI and formation of the SAM.

Proteins in aqueous solution are detectable by the sensor of the present invention. Proteins in aqueous solution are polyelectrolytes whose charges are dependent on the intrinsic isoelectric point of the protein as well as the ionic composition of the solution. Charged protein adsorption into geometrically complementary cavities of the thiol layer yields noticeable electric potential differences due to gold's conductive properties. An effective electrochemical biosensor capable of identifying specific protein concentrations, selectively sensing proteins, and in vitro protein detection is engineered as described below via use of gold-thiol SAMs.

Identification of the fibrinogen concentration in the blood stream is critical for early diagnosis of conditions such as coronary heart disease and cardiovascular disease. Accordingly, due to the extensive role in blood clotting, cellular and matrix interactions, and wound healing, fibrinogen, a glycoprotein and coagulation factor, was identified for protein detection. In a preferred embodiment, gold-thiol SAM was imprinted with fibrinogen to engineer a biosensor capable of identifying specific fibrinogen concentration in the bloodstream through voltage response testing. Similarly, hemoglobin, an iron-containing globulin protein critical for transporting oxygen throughout the bloodstream, can lead to anemia and iron deficiency when present in reduced levels in the blood. To demonstrate selectivity of the two-dimensional biosensor of the present invention, a fibrinogen imprinted chip was cross-tested with hemoglobin, fibrinogen, both proteins, and vice versa for a hemoglobin imprinted chip. While the two-dimensional SAM biosensor has been extensively studied for detection of biomolecules, little experimentation has been conducted for early diagnostic sensing of imprinted pathogens and bacteria.

Conventional methods of testing for bacteria with the SAM often involve imprinting with biomarkers rather than the bacteria itself. For example, Zhou, Y., et al., Potentiomeuϊc Sensor For Dipicolinic Acid, Biosensors and Bioelctronics, 20: 1851-1855 (2005), tested for the presence of anthrax using surfaces imprinted with dipicolinic acid, a biomarker for Bacillus anthracis and component of bacterial endospores. However, conventional methods fail to provide the bacterial biosensor for salivary testing, as provided in a preferred embodiment of the present invention.

In the present invention, a technique of imprinting the gold-thiol SAM with bacterial cells themselves is proposed, allowing for more precise detection of pathogens based on cell shape, conformation, and extracellular matrix (ECM) composition.

Pseudomonas aeruginosa, a Gram-negative rod-shaped Gammaproteobacteria, is an opportunistic pathogen responsible for serious infections in hospitalized patients with cancer, cystic fibrosis, and severe burns. Detection of this pathogen in such patients can provide doctors with a valuable early diagnosis of infection of the pulmonary and urinary tracts of immunocompromised individuals. Current detection of P. aeruginosa, however, relies on a tedious and expensive ELISA for ImmunoGlobulin G (IgG) antibodies. Instead, the present invention refines diagnostic methods for P. aeruginosa by imprinting the inexpensive gold SAM sensor with the bacteria. At the same time, Staphylococcus aureus, a Gram-positive coccus, is the cause for numerous skin infections and certain antibiotic-resistant strains, such as Methicillin-resistant S. aureus (MRSA), which can lead to widespread infection of vital organs. The sensor of the present invention utilizes selectivity of surface imprinted cavities in both P. aeruginosa and S. aureus. In order to simulate testing conditions of the body more practically, the sensor of the present invention recognizes bacteria in infected saliva samples. Saliva testing is becoming an increasingly popular as a medium for a noninvasive, time efficient testing, allowing for ready collection and requiring samples sizes of only 1-5 mL. As a fluid commonly excreted from the oral cavity, saliva represents an ideal environment for detection of most bacterial pathogens. Nanotechnology is a constantly growing field of science because of the numerous properties that emerge in particles on the nanoscale. However, it is important to consider long- term health effects of these nanoparticles. Human dermal fibroblasts represent an ideal cell type for testing the effects of nanoparticle exposure as these cells comprise the skin, the body's first barrier to environmental agents containing nanoparticles. Previous research has shown that intracellular presence of Au nanoparticles leads to alterations in cell spreading, adhesion, and growth. These nanoparticles cause extensive damage to actin fibrils of the cell cytoskeleton which is then transmitted to the ECM by integrin receptors. Fig. 2 shows interconnectedness of cell cytoskeleton and ECM, as described by Pernodet, N., et al., Adverse Effects of Citrate/Gold Nanoparticles on Human Dermal Fibroblasts, Small; 6: 766-773 (2006). Consequently, secretion of fibronectin, an ECM glycoprotein involved in cell adhesion, growth, and migration, as well as that of fibrinogen, another ECM protein, is diminished in human dermal fibroblasts exposed to Au nanoparticles, detectable by the two-dimensional protein biosensor of the present invention.

In parallel, the two-dimensional protein sensor of the present invention was applied to detect the point of differentiation of DPSC into osteoblasts. Stem cells demonstrate the unique ability to proliferate rapidly while differentiating into a specific cell lineage, which consequently make them a desirable tool for tissue repair. DPSC, in particular, are ideal stem cells because of the accessibility of their collection site, efficiency of cell extraction from the pulp tissue, high level of differentiability, and their demonstrated interactivity with biomaterials needed for tissue reconstruction. Multipotent DPSC can be differentiated into osteoblasts and odontoblasts by culturing cells with soluble stimuli in the induction media, such as the synthetic glucocorticoid Dexamethasone. Osteogenic differentiation of DPSC can be used for regenerative pulp treatment by accessing reparative dentin secreted from fully differentiated odontoblasts. However, chemically induced differentiation is unfavorable for eventual in vivo application of DPSC in the body for bone and tooth regeneration. Alternatively, differentiation of Mesenchymal Stem Cells (MSC) has been shown to be highly sensitive to tissue-level elasticity of the cell's microenvironment. Cell growth on soft matrices of elastic modulus 0.1 1.0 kPa yielded neurogenic cells, whereas MSC grown on rigid matrices of elastic modulus about 100 kPa, similar to that of collagenous bone, led to osteogenic differentiation. It has been hypothesized that the MSC sense the elasticity of the scaffold by pulling against the matrix and transducing cellular signals based on the force needed by the cell to deform the matrix. Once DPSC is differentiated into osteoblasts, calcium binding proteins is produced, such as osteocalcin, and can nucleate hydroxyapatite crystal formation in the ECM. In this sense, osteocalcin can serve as a protein biomarker for osteoblast formation. A preferred embodiment of the present invention plates DPSC on different thicknesses of polymer films and testing media samples for osteocalcin production with an imprinted two-dimensional biosensor. Consequently, monitoring is provided of DPSC stem cell differentiation into osteoblasts over extended periods of time.

To assess the viability of the SAM potenliometric biosensor as an effective tool for molecular detection, five procedures were performed: 1) building the biosensor and understanding SAM surface mechanics, 2) determining specific protein concentration and sensor selectivity, 3) salivary testing for bacterial pathogens, 4) In vitro sensing of damaged tissue, 5) In vitro sensing of adult stem cell differentiation. Experimental results obtained from the fabricated sensor are outlined in Table 1 from the two-dimensional gold-thiol SAM biosensor. Table 1

To develop an electrochemical biodetection system of the present invention that translates molecular presence into electric voltage readings, silicon (Si) wafers were plated with gold through chemical vapor deposition. In order to deposit the gold, Si wafers were placed in a vacuum chamber for twenty-four hours. An electric potential of 10,000 V was applied to sublimate the gold, which was then deposited onto the Si wafers using a vacuum again after the 24 hour period. The gold plates constructed through this process were then cut into chips of size 0.5 cm x 2.0 cm, which were cleaned by rinsing with de-ionized (DI) water and dried with nitrogen gas.

For each biosensing experiment involving the gold-thiol SAM, a chip was incubated at 22°C in a 10 mL solution of 2% thiol (Aldrich, 447528, 1 1 mercapto 1 undecanol 97%) and a specific molecule concentration for two hours. During incubation, thiol molecules formed a molecular monolayer atop the gold through sulfur-metal bonds, while the molecule became embedded in the thiol through hydrophihc interactions with the thiol's hydroxyl group. The chip was then removed from solution washed thoroughly with DI water for one minute to remove template molecules, leaving behind the geometrically complementary cavities shown in Fig. 3.

At this point the chip was ready for testing, and the template molecule was reintroduced in known concentrations. Voltage readings were taken from an electrochemical potentiometer (Orion 920A+ Multi-Channel Benchtop Meter) connected to an AgCl reference electrode bulb (Bioanalytical, BAS, Model MR-5275) and sensing electrode gold electrode 101, i.e. gold chip, and reference electrode 103, as shown in Fig. 4. Both electrodes were suspended in ten mL of Phosphate Buffered Saline (PBS) 107 (Dulbecco's Phosphate Buffered Saline: GIBCO, 14190144, Ix), and a magnetic stir bar 109 was introduced in the beaker atop a stir pad Potentiometer (Precision Scientific Co., MAGMTX, 65904, 50/60 eye) to increase the rate of molecular readsorption. Prior to the onset of testing, the potentiometer was calibrated using buffer solutions at 4.01, 7.00, and 10.01 pH. When testing with the protein, electric potential differences were recorded and calculated for AE=E- Eo, where E is the potential at a given molecular concentration and Eois the potential before testing with the molecule.

Imaging of the SAM was conducted to observe imprints and readsorption visually. A two-dimensional sensor was constructed for bacterial detection by incubating the gold chip in P. aeruginosa in water and thiol dissolved in ethanol, according to specifications shown in Table 1.

An Atomic Force Microscope (AFM) was used to image the topography of the SAM, allowing for nanoscale identification of the thiol, gold, and bacteria. The AFM used was a Digital Instruments Nanoscope IHA placed in a sealed glove box. The following three samples were imaged: 1 ) gold wafer, 2) gold wafer incubated in thiol only, 3) gold wafer incubated in thiol and P. aeruginosa before washing away bacteria. Samples were dried with nitrogen gas at 1 psi. Initial testing was conducted in contact mode, by which the AFM etched silicon cantilever was dragged laterally across the surface of the chip. However, this resulted in disruption of the composition of the monolayer and reduction in image resolution due to damage from frictional movement across the thiol layer. Subsequent tests were conducted using the AFM tapping mode by which the cantilever oscillated vertically atop the surface of the chip. Root Mean Square (RMS) roughness values were additionally calculated for each of the chips as an indication of overall roughness of the surface.

Further imaging of the SAM was performed using a Gemini 1530 (Zeiss) Scanning Electron Microscope (SEM) at magnifications of 6,00Ox and 30,000x. This technique of imaging provided visual confirmation of the formation of bacterial imprints by scanning the SAM surface with a high-energy electron beam. Following drying with nitrogen gas, each chip intended for testing was adhered to a flat aluminum specimen stub using pieces of conductive tape. The stub was then placed in the sample vacuum chamber of the SEM and left to rest for nine minutes. At this point, the electron beam was lowered over the surface for testing. All three chip setups used for the AFM were examined under the SEM in addition to a fourth gold wafer incubated in thiol and P. aeruginosa after washing away bacteria.

Specific protein concentration was then assessed and sensor selectivity determined. Voltage readings were obtained in terms of specific protein concentration, chips were tested and imprinted with the fibrinogen protein. A 10 mL incubation solution of 6% fibrinogen (Sigma Aldrich, F-2629, Fraction 1 from pig plasma) and 2% thiol was used for templating the chip with fibrinogen imprints. Fibrinogen in the incubation solution was dissolved in DI water at 1 mg/mL and thiol in ethanol at 0.4 mg/mL (Pharmco Aaper, 200 proof, absolute anhydrous, ACS/USP grade, 1 1 1000200). When testing the chip, fibrinogen dissolved in PBS was added to the testing beaker until reaching a fibrinogen concentration equal to that in the incubation solution. Voltage differences from the initial reading were measured while increasing fibrinogen concentration in the testing beaker. A standard linear curve of fibrinogen concentration vs. change in electric potential was developed. The accuracy of this linear regression was evaluated by testing the deviation of the curve from voltage responses to variable fibrinogen concentrations.

To evaluate the biosensor in terms of selectivity, the chip was cross-tested in a combination of fibrinogen and hemoglobin. Three gold-plated silicon chips were imprinted in 10 mL solutions of 6% fibrinogen, 2% thiol, and three other chips were imprinted in 10 mL solutions of 3% hemoglobin (Sigma Aldrich, CAS# 9008-02-0, Lyophilized powder, from bovine blood), 2% thiol. Each of the sets of three chips was exposed to three testing solutions: fibrinogen, hemoglobin, and a 1 : 1 combination of fibrinogen and hemoglobin. Additionally, two control chips were incubated with no proteins and tested separately for just fibrinogen or hemoglobin. Voltage differences from the initial reading were measured while increasing protein concentration in the testing beaker until the chip's imprints had saturated and voltage readings remained constant.

To demonstrate the two-dimensional sensor's potential for bacterial detection, particularly salivary testing for bacterial pathogens, the gold chip was first incubated in P. aeniginosa in DI water and thiol dissolved in ethanol. All bacterial concentrations were calculated using a solution of known concentration of approximately 10" bacteria/mL water. The 10 mL chip incubation solution consisted of 25 bacteria/mL and 2% thiol. After two hours of incubation at 22⁰C and washing of the chip, voltage responses were taken while increasing bacteria concentration in PBS in the testing beaker. A standard linear curve of/*, aeruginosa concentration vs. change in electric potential was then developed.

The biosensor was again tested for selectivity by cross testing the chip in a combination of P. aeruginosa and S. aureus. Three chips were imprinted in P. aeniginosa in 10 mL solutions of 25 and 2% thiol, and three other chips were imprinted S. aureus in 10 mL solutions of 25 bacteria/mL and 2% thiol. Each of the sets of three chips was exposed to three testing solutions in PBS: P. aeniginosa, S. aureus, and a 1:1 combination of both bacteria. Two additional control chips were incubated with no bacteria and tested individually for P. aeniginosa and S. aureus. Voltage readings were again taken from the potentiometer to test the sensor's selective response to differently shaped bacteria.

To assess the biosensor's sensitivity in a more realistic setting, tests were then taken with samples of the experimenter's own saliva. The 5 mL testing solution consisted of 1 mL human saliva and bacteria at a concentration of 43 bacteria/mL PBS. Three testing solutions were employed: no bacteria, P. aeruginosa, and a 1 : 1 combination of both bacteria. Each testing solution was kept for two hours in a 37°C incubation chamber to allow bacteria to replicate within the saliva. Three chips were imprinted with P. aeruginosa in 10 mL solutions of 25 bacteria/mL and 2% thiol. These imprinting solutions were incubated for two hours at 22°C. Voltage readings were taken until the chip's imprints had fully saturated.

Testing was performed of in vitro sensing of damaged tissue, and the efficacy of the biosensor in vitro required an appropriate setup with cells and the nanoparticles intended for exposure. The effects of Au-citrate nanoparticle exposure on human dermal fibroblast viability were assessed by plating cells as follows: cells (control), cells with 13nm diameter particles, cells with 45nm diameter particles. CF-29 human dermal fibroblasts from the established ATCC cell line were plated at 20,000 cells per well and treated with 500 μl_ of a designated concentration of citrate-stabilized Au nanoparticles in 2 niL of McCoy's media. Nanoparticle solutions for treating cells had concentrations of 130.29 μg/mL for 45nm particles and 948.5 μg/mL for 13nm particles. Cells were treated with nanoparticles 3 days after plating. Additionally, samples of media were taken and replaced in each well for testing ECM protein concentrations using the two-dimensional SAM biosensor on days 3, 5, and 7.

Au-citrate nanoparticles, which are known to be cytotoxic to human dermal fibroblasts, were synthesized as outlined by Pernodet (2006). Citric acid acted as a stabilizer, preventing gold from aggregating. Large nanoparticles of 45 nm diameter were produced by first setting a three- necked flask under a condenser and boiling 25 mg of KAuCI₄ in 98 niL DI water. 2.5 mL of a 1.5% sodium-citrate solution was added under vigorous stirring, causing a color transformation from gray to purple. Small nanoparticles oΩ3 nm diameter were synthesized by heating 0.1 mL HAuCU in 95 mL DI water to its boiling point. 200 mg of sodium citrate dissolved in 5 mL DI water were then added, likewise producing a color change from yellow to gray to dark red. Finally, the solution was gently boiled for 40-50 minutes and then slowly cooled down. After exposure of cells to nanoparticles, cell media samples were tested on the biosensor for ECM proteins fibronectin and fibrinogen. In order to test for these proteins, gold chips were incubated in separate solutions of 2% fibronectin, 2% thiol and 6% fibrinogen, 2% thiol at 22°C.

In vitro sensing of adult stem cell differentiation was performed. To assess the differentiation of DPSC into osteoblast-like cells and the consequent production of osteocalcin, cells were plated on various polymer film surfaces comprised of polystyrene (PS), polybutadiene (PB), and a 65%-35% mixture of PS to PB. Polymer films were produced by dissolving PS, PB, and the PS-PB copolymer in toluene at 3 mg/mL for thin films and 20 mg/mL for thick films. These solutions were spun cast onto 1 cm² Si wafers using a Headway Research™ spin-caster at 2500 rpm for 30 seconds. The solvent evaporated forming a uniformly distributed thin film of the desired polymer on the wafer surface. Film thicknesses were calculated using a Rudolph Research™ ellipsometer to be approximately 200 A for all thin films and 2000 A for all thick films.

Dental pulp stem cells from the third molar teeth of a 21 -year-old male were plated at 5,000 cells per well. DPSC were arranged as follows: plastic (+induction), plastic (-induction), thin PB, thick PB, thin PS, thick PS, thin PS/PB, thick PS/PB. Cells were plated in a 24 well plate with each well containing 1 mL of MEM-a media (Invitrogen, cat # A 10490-01 ) supplemented with 10% Fetal Bovine Serum, 100 μM 1-ascorbic acid 2-phosphate, 2 mM I- glutamine, 100 units/ml penicillin/100 μg/ml streptomycin, and 2.9 mmol/L inorganic phosphate. Chemical osteogenic induction for cells plated on plastic required an additional 10^'s mol/'L dexamethasone (Sigma-Aldrich, D4902) in the media. The plastic used for plating these cells was tissue culture plastic (Nunc T- 175, T-75, P-100) made of sulfonated polystyrene treated with gas plasma. For all eight setups, 100 μL of a cell solution diluted to 50,000 cells/mL media was added to each of the 24 wells or the 1 cm² polymer film substrate. Cell media samples were extracted for testing and replaced in each setup on days 3, 5, and 7. Gold chips were incubated in separate solutions 0.02% osteocalcin (Gen Way, purified bovine native protein, cat# 1 1-51 1 - 248795) and 2% thiol in order to test for osteocalcin in the media samples.

The results of the imaging microscopy conducted on gold-thiol assemblies before and after washing in order to better understand the mechanics of two-dimensional MI on the SAM are as follows. Atomic force microscopy (AFM) provided a detailed topographical analysis of the texture of the SAM surface. Imaging of just the gold chip revealed minor defects and impurities in the deposited gold, as previously characterized by Love, C. et al., Self-Assembled Monolayers of Thiolates on Metals us a Form ofNunotechnology, Chem. Rev.; 105: 1 103-1 169 (2005). Imaging of the gold-thiol layer was able to distinguish individually dispersed thiol molecules at higher altitudes than the gold, indications of the formation of a monolayer. Analysis of the bacteria-imprinted gold chip prior to AFM imaging of the surface of the gold chip prior to incubation, after incubation with thiol, and after incubation with thiol and bacteria, when unwashed. Scan rates and sizes used to generate the image are included.

Washing indicated that bacteria had effectively adsorbed to the SAM surface. The limited presence of bacteria in AFM scans can be explained by the relatively low bacterial concentration with which the chip was incubated -25 bacteria/niL. RMS roughness values were calculated from AFM imaging of the three surfaces at a scan size of 6 urn and reported in Table 2, showing RMS roughness values from the three AFM chip setups.

Table 2

Ideal for quantifying change in surface topography, RMS roughness (S_q) represents the standard deviation from the median surface level, u, across coordinate pixel locations (x, y) and total number of data points, MN. The value can be modeled by Equation (2):

The presence of an unevenly dispersed monolayer on the gold accounted for gold-thiol's increased roughness. The chip incubated with bacteria prior to washing had the highest roughness measurements as Equation (2). An SEM was used to identify monolayer formation and bacterial adsorption to the SAM as shown in Fig. 5. Imaging of only the gold deposited wafer showed that this surface was clean prior to incubation. The gold chip incubated with only thiol produced a lighter image as the organic films appear transparent under the SEM. Individual thiol molecules were not entirely discernable at this magnification but their general presence is confirmed by lighter areas dispersed throughout the entire surface. Imaging of the bacteria-incubated SAM prior to washing depicted bacterial adsorption and confirmed specific characteristics of P. aeruginosa, such as its rod shape and the presence of pili. Imprints left after washing were visible as faded outlines of the bacteria and possible protein residue left from bacteria. Additionally, these imprints appear darker, indicating the reduced presence of thiol. These results are important because they confirm the formation of the SAM, adsorption of bacteria onto the surface, and remnants of geometrically complementary imprints on the chip surface.

Specific protein concentration and sensor selectivity were determined as follows. To interpret voltage readings in terms of protein concentration, a standard curve of voltage difference as a function of fibrinogen testing concentration was constructed. Initial testing indicated the maximum protein concentration for a 6% fibrinogen-imprinted chip at which the curve remained linear in shape was approximately 17.5 μg/mL fibrinogen. Consequently, the linear regression Equation (3): y=1.288x + 3.667, (3) with voltage difference in mV, y, and concentration in μg/mL, x, for the standard curve of fibrinogen was used to calculate the concentration of unidentified solutions, assuming a continuous linear relationship at all fibrinogen concentrations below 17.5 μg/mL. To assess the accuracy of the standard curve, a variable point test of fibrinogen concentrations 4.79 and 9.54 μg/mL was conducted and resulting voltage readings were compared to the standard curve. As shown in Fig. 6, both voltage differences from these fibrinogen solutions were within the error bars of the curve, indicating that these values were within one standard deviation of the curve and confirming that this line is an accurate representation of fibrinogen concentration in solution.

To demonstrate the selective capacity of the biosensor for protein detection, voltage readings of fibrinogen and hemoglobin imprinted chips were taken in various combinations of the two proteins. Fig. 7 shows voltage responses for fibrinogen-imprinted chips. The sensor tested with hemoglobin gave off the lowest potential differences, other than the control with no protein imprints, because cavities on this chip were complementary to the shape of the fibrinogen protein, preventing hemoglobin from binding effectively. However, the fibrinogen chip in a testing solution of both gave the highest response as charged hemoglobin proteins landed in imprints intended for fibrinogen, consequently producing higher voltage readings than would occur from adsorption of only the uncharged fibrinogen molecule. As shown in Fig. 7, testing with the opposite protein again yielded the lowest voltage response, as these proteins could not fit into differently shaped cavities. Unlike with the fibrinogen imprinted chip, testing with a combination of the proteins did not produce the highest potential readings as this caused the uncharged fibrinogen to occupy cavities intended for charged hemoglobin that would more greatly increase the voltage across the chip. These results are critical because they indicate that the two-dimensional SAM chip can be used as an accurate indicator of protein concentration and demonstrate the selectivity of the sensor for protein detection.

Salivary testing was performed for bacterial pathogens. To assess the applicability of the two-dimensional biosensor to bacterial detection, a standard curve of electric potential response to P. aeruginosa concentration was constructed. Preliminary tests of a chip incubated in 25 bacteria/mL indicated that the maximum concentration at which the curve was linear was approximately 21 bacteria/mL. The linear regression equation for this curve of f. aeruginosa, is provided in Equation (4): y=0.1013x + 1.4997, (4) with voltage difference in mV, y, and number of bacteria added, x; and an R² value of 0.9646 used to calculate the concentration of unidentified solutions for P. aeruginosa solutions of concentration below 21 bacteria/mL. Similar tests of biosensor selectivity were conducted by cross testing with both P. aeruginosa and S. aurenis. Figs. 9 and 10 show voltage responses for P. aeruginosa and S. aureus imprinted chips, respectively. For both scenarios, chips with no imprints yielded the lowest response for testing due to the absence of imprint cavities for bacterial adsorption. Testing with the opposite bacteria in both setups yielded the second lowest response as these imprint cavities were not intended for these bacteria. The highest response was present in testing with just the intended bacteria, which could appropriately readsorb to complementary cavities. The combination yielded the second highest response, as the opposite bacteria impeded movement of templated bacteria to their complementary cavities. To demonstrate a practical testing solution, bacterial tests with P. aeruginosa were also conducted in infected samples of our own saliva. As expected, testing without bacteria (control) resulted in the lowest voltage response while testing. Fig. 1 1 is a graph showing potentiometric response of P. aeruginosa-imprϊnied, with only

P. aeruginosa being most responsive. In Fig. 11, potentiometric response of P. aentginosa- imprinted with only P. aeruginosa was most responsive, consistent with results from tests conducted in PBS. Not only do these results verify the accuracy and selectivity of our novel application of the biosensor to bacterial detection, but also indicate that saliva is an acceptable diagnostic medium for practical bacterial biosensing.

In Vitro sensing of damaged tissue. To assess cellular damage from nanoparticle exposure, tests of protein secretion in the ECM were conducted as it was hypothesized that internal cytoskeletal damage of actin fibrils would be relayed to the ECM by means of integrin receptors in the membrane. Fibronectin and fibrinogen secretion were highest in cells with no nanoparticle exposure, as shown in Figs. 12 and 13, respectively. Control cells continued to produce more of the protein over time in the absence of cytoskeletal damage. However, cells exposed to the larger 45 nm Au particles experienced the least ECM protein secretion as these particles caused more significant damage to actin fibrils than did smaller 13 nm particles. Furthermore, protein secretion for nanoparticle exposed cells continually decreased over the time period as existing nanoparticles caused further damage to cells. The results of this experiment confirm the hypothesis that known cytoskeletal damage due to Au nanoparticles can lead to a reduction in ECM protein secretion. //; Vitro sensing of adult stem cell differentiation. Osteogenic stem cell differentiation of

DPSC was analyzed in terms of the biosensor's detection of osteocalcin, a protein involved in biomineralization. Induction was attempted through chemical means with dexamethasone as well as through surface interactions by growing cells on polymer films of varying hardness. Results throughout a seven-day period indicated a steady increase in osteocalcin secretion, confirming differentiation in all setups with the exception of control cells plated on plastic with no induction factors in the media. Cells plated on plastic and exposed to dexamethasone consistently produced the highest detectable readings of osteocalcin, indicating a most effective chemical induction of all setups for osteogenic differentiation. At the same time, attempts at differentiation through substrate mechanics, a biologically safer alternative, confirmed the hypothesis that DPSC grown on harder surfaces similar to the rigid surface characterization of the collagenous bone would lead to further osteogenic differentiation and greater osteocalcin production than cells grown on softer substrates. For the polymer substrates of PS, PB, and PS-PB copolymer, the thin film surface always led to greater osteocalcin secretion than the thick film surface. These results are expected because thin polymer films tend to be stiffer, allowing for little flexibility from the Si wafer. Films made from PS led to the greatest differentiation, followed by the copolymer blend. The tendency for greater differentiation on PS than on PB is consistent with the mechanical characteristics of the hard PS versus those of the synthetic rubber PB polymer. The results of the osteocalcin testing are critical in that the biosensor can be applied for in vitro detection of stem cell differentiation into osteoblast-like cells and serve as an indicator of bone formation and biomineralization.

The two-dimensional molecular imprinting can, in a preferred embodiment, be utilized to construct an electrochemical biosensor diagnostic tool. In regard to the mechanics of the bacterial biosensor, SEM and AFM imaging were used to develop a topographical map of the SAM surface and to calculate roughness measurements. Selectivity and sensitivity of this instrument for protein detection by cross testing the sensor with both fibrinogen and hemoglobin proved that a protein-imprinted sensor could selectively distinguish the two proteins in solution, and that the sensor provides an accurate measure of protein concentration in unknown solutions. Accordingly, the present invention provides a novel method for bacterial pathogen detection through molecular imprinting, which can be utilized on human saliva samples.

The protein sensor can be applied to an in vitro setting, as shown by cell media sample testing. The detected decrease in secretion of ECM glycoproteins fibrinogen and fibronectin from Au nanoparticle exposed human dermal fibroblasts supports the hypothesis that internal damage to the cytoskeleton from nanoparticle presence was relayed to the ECM. Additionally, the sensed increase in expression of osteocalcin in DPSC grown on thin hard films of PS, PB, and the copolymer blend indicate the osteogenic differentiation of these cells in the absence of chemical inducers. The sensor can be applied for bone detection and as an indicator of tissue damage from nanoparticle exposure. The present approach has also been applied successfully to CarcinoEmbryonic Antigen

(CEA), amylase and also to poliovirus, thereby showing that the protein sensor can be applied in a general detection method. In a preferred embodiment, /he sensing element consists of a gold- coated silicon chip onto which alkanethiol molecules with hydroxyl end groups are chemically bound through a sulfur-metal bond; via self-assembly that produces a well-organized monolayer. If the target molecule, i.e. template, is present when the monolayer forms, the target molecule creates an imprint in the SAM matrix. Removal of the template molecules from the surface leaves behind cavities having a size, shape and hydrphobicity complementary only with the template molecules. This complementarity provides the high affinity to the template molecules and its specificity. To assay for a molecule, the sensing element is attached to a potentiometer. When a biological molecule that is charged in aqueous solutions binds onto a complementary cavity of the sensing element, the potential of the sensing element changes, which is measured potentiometrically against a reference electrode. As demonstrated here, the present method is suitable for the detection of cellular proteins, including biomarkers, and of supramolecular protein assemblies, such as the capsid of virions. In a preferred embodiment, materials of 1 1 -mercapto- 1 -undecanol, CEA. human α- amylase, bovine hemoglobin and routine chemical reagents were from Sigma, St Louis, MO and fetal bovine serum from Hyclone, Logan, UT, and a sensing element was prepared as follows.

Gold (5OθA) coated silicon substrates (I^χ2cm^~) were cleaned with de-ionized water and dried under nitrogen gas. Blend solutions were obtained as follows: The template (proteins or viruses) were dissolved de-ionized water and the alkanethiols were dissolved in acetic acid, and the two solutions were mixed (19/1 \v/v\, respectively) to 10^"4 M final thiol concentration. To avoid aggregation of the templating molecules, we chose low concentration: 0.644 nM for poliovirus, 0.833 nM for CEA, representing 3.88x 10¹ ' and 5.O2χ lθ" molecules/mL, respectively, and 200 ng protein/mL for amylase (for poliovirus, the pH of the solution was neutral). The gold-coated electrode was immersed into the blend solution at room temperature for more than two hours, and rinsed with running de-ionized water for two minutes to remove the template. Electrodes were stored in ambient conditions and their sensitivity was assessed within eighteen hours. The control electrode was prepared by immersing it into the thiol solution, without template solution.

A cell culture of LoVo human colorectal cancer cell line (ATCC, Manassas, VA) was grown as described in Ham's F-12K medium (Cellgro) supplemented with 10% fetal bovine serum. CEA was measured in media using CEA ELISA Test Kit (BioCheck, Foster City, CA).

Electrochemical Measurements were obtained as follows. Polio virus was assayed in 8 mL Dulbecco's phosphate buffered saline (DPBS: 2.67mM KCl, 8.06 mM Na₂HPO₄(H₂O)₇, 1.47 mM KH₂PO₄, 137.93 mM NaCI; pH=7.15), CEA in culture medium (pH~7.0), amylase in 10 mL FBS or DPBS in a beaker with a magnetic stirrer. A two-electrode system was used: the Ag/ AgCl reference electrode and the template/thiol modified (or control) sensor. The potential between test and reference electrodes was measured with a potentiometer (Orion 920). The sensing signal (ΔE) was the potential change of the test electrode after target molecule addition (E) compared to baseline (Eo): ΔE = E - Eo.

In the present embodiment, detection of was performed of clinically important proteins including the cancer biomarker CEA and the pancreatic enzyme amylase as well as of poliovirus, the virus that causes poliomyelitis. CEA is used predominantly as a marker for colorectal cancer, often to monitor tumor recurrence following its treatment. CEA, a large glycoprotein, has a complex structure with carbohydrate side chains; its molecular weight is 180 -200 kDa and its X- ray radius of gyration is 8.0 nm. In normal adults serum CEA is less than 2.5 ng/niL for non- smokers and less than 5.0 ng/mL for smokers. In the appropriate clinical setting, CEA levels >5 times the upper limit of normal suggest the presence of colorectal cancer, whereas higher levels are usually associated with metastatic cancer. The poliovirus is a small non-enveloped positive strand RNA virus. The virion of the poliovirus consists of a single RNA molecule surrounded by an icosahedral protein capsid (60 copies of each of the VPl, VP2, VP3 and VP4 proteins). The diameter of the virus particle is 20-30 nm and its molecular weight is ~8.3xl O⁶ Da. Poliovirus was studied to confirm that the method is sensitive to large, complex molecules of clinical importance.

The ability of the CEA sensor to detect CEA, either purified or in the culture medium of LoVo human colorectal cancer cells, which are known to secrete CEA was confirmed as follows. The potential ΔE recorded by the CEA imprinted electrode changed in response to stepwise additions (2.5 ng/mL) of CEA dissolved in Ham's F-12K medium supplemented with 10% fetal bovine serum. An initial steep increase in ΔE at protein concentrations up to -75 ng/mL was followed by a more gradual response up to -250 ng/mL. Chip to chip reproducibility was tested and the average of the coefficient of variation for all the data points was 17.7%. An evaluation of specificity of the response was performed in three ways. First, the CEA imprinted electrode was exposed to a solution of bovine hemoglobin, whose concentration was increased, as for CEA, stepwise from 2.5 to 250 ng/mL. The ΔE remained essentially unchanged. Second, a control electrode (not imprinted with any protein) gave no response when exposed to CEA solutions. Third, a sensor imprinted with hemoglobin gave no response when exposed to incremental CEA solutions applied as described above.

The sensor was then employed to assay CEA levels in the culture medium of LoVo human colon cancer cells. The assay was performed in a similar fashion. Similar to the previous findings, the ΔE recorded by the CEA imprinted electrode increased rapidly at low volumes of culture medium and appeared to reach a plateau at higher volumes, indicating a clear concentration-dependent response. Culture medium was assayed from cells incubated for 8, 18 and 24 hours, showing a saturation value increase with increasing incubation times.

Using a concentration response curve based on known CEA concentrations to calibrate the sensor, a CEA concentration in the LoVo cell medium was determined to increase in parallel with the incubation time of the cells. As validation, the CEA concentration in the cell culture medium was measured independently by an enzyme immunoassay method, and an excellent agreement of results was obtained.

Finally, the incubation time of the cells was kept the same (20 hours), but density was varied (0.9, 2.6 and 4.8x 10⁶ cells/plate). The CEA values obtained from the CEA sensor and the immunoassay were found to be in excellent agreement. Thus, the SMI Self- Assembled Monolayer (SAM) method successfully assays CEA over a wide range of concentrations with accuracy essentially identical to that of immunoassay methods.

Detection of amylase. The performance of the method for proteins other than CEA was tested using a sensor for pancreatic amylase. The sensor was fabricated by imprinting with α- amylase from human pancreas. To test its performance, we assayed as previously incremental volumes of a solution of purified α-amylase in fetal bovine serum (FBS); the highest α-amylase concentration tested was 300 ng/mL. The type of ΔE response was similar to that with CEA: following an initial steep rise, with the ΔE response had a much lower slope, reaching essentially a plateau. Of note, the sensor responded weakly (and negatively) to the addition of FBS dissolved in DPBS. Since FBS contains a large number of proteins, our results establish the specificity of the sensor and its ability to operate efficiently in multi-component protein solutions.

Detection of poliovirus. To determine whether the present method can assay larger and more complex molecules, we focused our efforts on the detection of poliovirus. The sensing electrode was templated in a dilute poliovirus/thiol solution and its response to incremental concentrations of virions was recorded as previously. As shown in Fig. 17, the initial rise in ΔE was followed by a plateau starting at about 4,200 ng/mL of virions. The specificity of the response was tested in two ways. First, the same electrode was exposed to adenovirus 5, which is a medium-sized (90-100 nm), nonenveloped icosahedral virus containing double-stranded DNA. There was practically no change in ΔE at viral concentrations identical to those used for the poliovirus. Second, a non-imprinted electrode was exposed to the poliovirus and, as shown, the ΔE remained virtually unchanged.

The findings demonstrate the application of the principles of molecular imprinting to the development of a new method for the detection of protein cancer biomarkers and to protein- based macromolecular structures such as a virion. Our data indicate that this approach has the potential of generating a general assay methodology that could be highly sensitive, specific, rapid, simple and likely inexpensive.

Two aspects of the inventive approach allow application as a general assay method: a) the SAM matrix we used allows our sensing element to interact with both hydrophobic and hydrophilic domains of proteins, and b) imprints from only a small portion of a large protein (at times far larger than the thickness of the entire imprinting SAM matrix) are sufficient for its detection by the sensor.

CEA and α-amylase, as well as the poliovirus have both hydrophobic and hydrophilic domains/regions in their outer surfaces. Given the obtained results, both hydrophobic and hydrophilic interactions with the sensor have likely occurred. Indeed, the chemical structure of the sensor allows for such interactions. The hydroxyl terminated alkanethiol SAM on the gold surface makes possible hydrogen bonding to the appropriate groups of amino acid residues from hydrophilic regions of the test protein. In addition, amino acid residues from hydrophobic regions, exemplified by the methionine (Met) residue, are adsorbed to the gold surface through hydrophobic interactions and/or electrostatic forces. The second interesting aspect of the method of the present invention is that it requires only a partial (in fact, very small) imprint on the sensor from the analyte. The thickness of SAM is around 1.5-2 nm, whereas the size — technically the radius of gyration on X-ray scattering — of CEA is -8 nm, and of poliovirus -20-30 nm. Thus only a small portion of the molecule can be in contact with the surface of the sensor.

The specificity of the sensor likely has two sources, steric specificity and chemical specificity. Steric specificity comes for the formation of the cavity perse. However, the orientation of surface functional groups adds the chemical specificity of the interaction between the analyte and the sensor. Such partial information, with respect to the much larger size of proteins or virions, is sufficient for analyte recognition.

The molecular structure of the sensor underlies both processes and makes possible the practical use of these sensors. The hydrogen bonding with the hydrophilic groups on the protein surface, as well as the electrostatic and hydrophobic interactions between the hydrophobic regions of the proteins and the gold surface, are sufficient to bind the target molecules from the solution, but also weaker than covalent bonds to allow their removal following the templating process. When the template molecules were adsorbed onto the electrode surface, the highly structured order of the surface amino acid groups were remembered by the combination of these interactions. At the same time, the template molecule distorted the planar symmetry of the SAM and introduced a gradient in the intermolecular positions and forces. This gradient was unique to the specific features on the imprinting molecule, and hence, in addition to the steric specificity, it generated chemical specificity during the imprinting process. The variation in bond strength has been elegantly demonstrated in measurements using nanoparticles where the radial position of the amide groups was varied. Removing the template, then leaves behind a templated cavity in the SAM, which maintains the shape of the molecule of the surface amino acid groups. Since this shape is at once specific and local, recognition of the analyte can be achieved even if only a small fraction of the protein surface presents at the templated electrodes. Therefore, MI is far more versatile than originally thought.

Data from the experiments utilizing the sensor of the present invention show that the use of water compatible hydroxyl terminated alkanethiol chains to produce the SAM extends the technique of SMI to big hydrophilic molecules. Consequently, the technique can now be used for sensing complex biological molecules such as cancer biomarkers and viruses, which are much larger than the thickness of the SAM matrix. The technique was applied to the detection of purified CEA molecules and those produced by living cells in culture. Calibration of the method indicated that quantities as small as 2.5ng/mL could be detected. Hence the production of the CEA as a function of cell number and incubation time could be tracked. An SMI fabricated amylase sensor demonstrated a good selectivity in the real serum test, without other proteins affecting its sensitivity. Finally, the detector was also shown to successfully detect small quantities of poliovirus where a robust signal was detected. Crosscheck demonstrated that the signal was not affected by the presence of other viruses, such as the adenovirus.

That is, data from experiments performed using the sensor of the present invention establish the principle and application of SMI to the detection of both proteins and virus. Using this methodology to the detection and quantification of cancer biomarkers may simplify current approaches. Configuring this method to simultaneously assay tens of cancer biomarkers, something entirely feasible, could greatly facilitate biomarker development and diagnostic use. Further work is needed to explore the range of diagnostic and other analytical applications of our novel method.

In a preferred embodiment, nanoparticle based biosensors and treatment methods are provided. In one aspect, the invention is directed to a cancer-treatment composition containing platinum nanoparticles (i.e., platinum nanoparticle cores) coated with FA molecules. As used herein, 'folic acid molecule' is used synonymously with 'folate.' Both 'folic acid' and 'folate' are meant to include not only the FA molecule, but also salts or chemical derivatives of FA that can also target folate receptors. The platinum (Pt) nanoparticle core preferably has a diameter of at least about one nanometer (nm) and up to about 15 nm, and more preferably up to about 10 or 12 nm. As used herein, the 'diameter' of the nanoparticle refers to the longest dimension of the nanoparticle and does not include coatings or molecular groups attached or associated with the surface of the nanoparticle. In particular embodiments, the diameter of the Pt nanoparticle core is no more than about 9 nm, or no more than about 8 nm, or no more than about 7 nm, or no more than about 6 nm, or no more than about 5 nm. In other embodiments, the Pt nanoparticle core has a diameter of at least about 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, or 9 nm. Any range derived from the foregoing minima and maxima are also applicable herein. For example, in different embodiments, the Pt nanoparticle core can have a diameter in the range of about 1-12 nm, 1- 10 nm, 2-10 nm, 3-10 nm, 4-10 nm, 5-10 nm, 6-10 nm, 7-10 nm, 8-10 nm, 9-10 nm, 1-8 nm, 2-8 nm, 3-8 nm, 4-8 nm, 5-8 nm, 6-8 nm, 7-8 nm, 1-5 nm, 2-5 nm, 3-5 nm, 4-5 nm, 1-4 nm, 2-4 nm, 3-4 nm, 1-3 nm, 2-3 nm, or 1 -2 nm. The Pt nanoparticle cores can be approximately monodisperse, and thus, essentially unvaried in size, or alternatively, polydisperse in size by any suitable degree.

The Pt nanoparticle core is typically substantially spheroidal in shape, but numerous other shapes are suitable. Some other suitable shapes include ovoid, cuboidal, polygonal (i.e., faceted), tubular, disc, prism, and amorphous.

The Pt nanoparticles can be synthesized according to any of the procedures well-known in the art for producing nanoparticles of zerovalent platinum. For example, the Pt nanoparticles can be fabricated by chemical reduction (e.g., aqueous-based reduction of a platinum salt or platinum complex by, for example, hydrogen, citrate, NaBH4, hydroxylamine, H₃PO₂, or other reducing agent), sonication, thermolysis, or Chemical Vapor Deposition (CVD).

The Pt nanoparticles can be functionalized with FA molecules by any suitable method. For example, in one embodiment, a platinum salt (e.g., K^PtCl₄ or a hexachloroplatinate or chloroplatinic acid) is reduclively treated in the presence of FA molecules such that the FA molecules form a coating on the surface of the Pt nanoparticles.

Typically, the Pt nanoparticles, either during or after their formation, are additionally treated with metal-binding molecules or polymers. When used during the reductive step, the metal-binding molecules or polymers typically function to stabilize the Pt nanoparticles during growth. As used herein, 'stabilizing' the Pt nanoparticles during growth means controlling the growth of Pt nanoparticles so that they do not agglomerate or grow beyond a desired size. The metal-binding molecules or polymers accomplish this by interacting with the surface of the Pt nanoparticles. The interaction can be covalent or non-covalent in nature. Typically, at least some portion of the metal-binding molecules or polymers remain on the Pt nanoparticle surface after the FA-Pt nanoparticle complex is isolated. The metal-binding molecules or polymers also very often function to alter the properties of the Pt nanoparticles to make them more suitable for administration to a mammal. For example, the metal-binding molecules or polymers can render the nanoparticles sufficiently hydrophilic (e.g., by incoφoration of hydrophilic groups, such as hydroxy or ethyleneoxy groups) or sufficiently selective or specific in their binding (i.e., such that non-specific binding is minimized).

Some examples of metal-binding molecules suitable for stabilizing or functionalizing Pt nanoparticles include sulfur-containing molecules (e.g., thiols, sulfides, and disulfides), amines (particularly primary amines), and phosphines (particularly organophosphines). The tripeptide glutathione is a particular example of a biocompatible thiol that may be used as a metal-binding molecule. The metal-binding molecules often form a Self-Assembled Monolayer (SAM) or multiple layer system. Some examples of metal-binding polymers suitable for stabilizing or functionalizing Pt nanoparticles include the dextrans (e.g., carboxymethyl dextran), dextroses, celluloses and their derivatives, polyethylene oxides or glycols (PEGs), albumin, and the like.

Metal-binding molecules can be made to bind to the surface of the Pt nanoparticle by any of the means known in the art to produce functionalized Pt nanoparticles suitable for ingestion by a subject. Typically, the metal-binding molecules are added before, during, or after formation of a dispersion of the Pt nanoparticles. The Pt nanoparticles can be dispersed in and reacted with the metal-binding molecules either in a hydrophilic solvent (e.g., aqueous or alcoholic), hydrophobic solvent, or a combination thereof. The functionalized Pt nanoparticles can be isolated by any suitable method known in the art, including, for example, precipitation, filtration, or adsorption.

In a particular embodiment, a metal-binding molecule or polymer interacting with the surface of the Pt nanoparticle is functionalized with one or more folate-reactive groups (e.g., amino, carboxy, epoxy, aldehyde, alkyl halide, isocyanate, hydrazido, or semicarbazide groups) such that the folate-reactive groups can react with and form a covalent bond with the FA molecule. In some embodiments, the reactive group is capable of binding to the FA molecule without being activated in some manner, whereas in other embodiments the reactive group is activated in order to bind to the FA molecule. Alternatively, a functional group of FA is activated or converted "by a linking group to a group capable of reacting with a group on the metal-binding molecule or polymer. In such an embodiment, the molecule or polymer becomes a linker between the platinum core and FA molecule such that the FA molecule is indirectly but covalently bound to the platinum core. For example, the Pt nanoparticles can be coated with carboxymethyl dextran to provide carboxy-functionality to the nanoparticles. Alternatively, carboxy-functionality can be provided to the nanoparticles by coating the nanoparticles with a polysaccharide and reacting the coating with a haloacetic acid under suitable conditions. In one embodiment, the carboxy functional group in the acid state (-COOH) is reacted with an amino group of an FA molecule to form a FA-Pt nanoparticle ionic complex.

In another embodiment, the carboxy functional group is reacted with an FA molecule by, for example, first converting the carboxy group to an activated ester (e.g., by N- hydroxysuccinimide (NHS) or carbodiimide (CDI)) and reacting this with the phenolic or amino group of the FA molecule. RN'-dicyclohexylcarbodiimide (DCC), and l-ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride (EDC) are often used in combination with NHS or sulfo-NHS. Alternatively, hydroxy or carboxy groups of a metal-binding polymer can be converted to other reactive groups, such as aldehyde groups (by appropriate chemical oxidation or reduction, respectively) or epoxy groups (e.g., by reaction with epichlorohydrin), all of which are reactive with an FA molecule under suitable conditions.

A functionalized metal-binding molecule or polymer can also be linked to FA by a secondary (i.e., intermediate) linker (coupler). For example, amino-amino coupling reagents can be employed to link an amino group of a metal-binding molecule or polymer with an amino group of the FA molecule. Some examples of suitable amino-amino coupling reagents include diisocyanates, alkyl dihalides, dialdehydes, disuccinimidyl suberate (DSS), disuccinimidyl tartrate (DST), and disulfosuccinimidyl tartrate (sulfo-DST), all of which are commercially available. Or, for example, amino-thiol coupling agents can be employed to link a thiol group of a metal-binding molecule or polymer with an amino group of the FA molecule. Some examples of suitable amino-thiol coupling reagents include succinimidyl 4-(N-maleimidomethyl)- cyclohexane-l-carboxylate (SMCC), and sulfosuccinimidyl 4-(N-maleimidomethyl)- cyclohexane-1 -carboxylatc (sulfo-SMCC).

The coupling agents, such as the ones described above, can be reacted with the Pt nanoparticle and FA molecule in any suitable manner that results in the FA molecule being attached, via the coupling agent, to the Pt nanoparticle. For example, by methods known in the art, functionalized Pt nanoparticlcs can be reacted to bind with one end of a coupling agent, and this subsequently reacted with FA. Alternatively, by methods known in the art, the FA molecule can be reacted to bind with one end of a coupling agent, and this subsequently reacted with the functionalized Pt nanoparticle. Other methods of coupling the FA molecule and Pt nanoparticle are possible and within the scope of the present invention.

In order to render the Pt nanoparticles biologically compatible and/or to prevent agglomeration, they are preferably hydrophilic to some degree. In one embodiment, the Pt nanoparticles are made sufficiently hydrophilic by incorporation of a sufficient quantity of FA molecules on their surfaces. In another embodiment, the Pt nanoparticles are made sufficiently hydrophilic by incorporation of a hydrophilic polymer, such as any of the hydrophilic polymers described above. In yet another embodiment, the Pt nanoparticles are made sufficiently hydrophilic by incorporation of hydrophilic non-reactive metal-binding molecules on their surfaces. An example of hydrophilic non-reactive metal binding molecules include those belonging to the class having the formula HS-R-(OH)_m, wherein m is 0 (i.e., -OH not present), 1, or any suitable integer, and R represents a carbon-containing group (for example, 1-18 carbon atoms), which can be saturated or unsaturated, and straight-chained, branched, or cyclic. The hydrophilic metal-binding molecules (i.e., the group R) can also contain any number of ethylene oxide (EO) groups, amine linkages, amide linkages, or sulfonate groups. Such hydrophilic groups also confer hydrophilicity and can be used along with or in the absence of hydroxy groups.

When reactive metal-binding molecules are used, they typically account for a portion (i.e., less than 100 mole percent) of the total amount of metal-binding molecules coating the surface of the Pt nanoparticle. The remainder of the metal-binding molecules do not possess a reactive group but can possess non-reactive functional groups. For example, the functionalized metal-binding molecules may comprise up to about 90, or 80, or 70, or 60, or 50, or 40, or 30, or 20, or 10, or 5, or 2, or 1 mole percent of the total amount of metal-binding molecules. The FA molecule can also be linked to the Pt nanoparticle by a coupling agent which contains a biological linking component. In one embodiment, the FA molecule can be linked to the platinum nanoparticle by making use of the well-known specific binding of biotin to avidin or streptavidin. For example, a biotinylation reagent can be reacted with a functionalized Pt nanoparticle to functional ize the nanoparticle with biotin. The biotin-functionalized Pt nanoparticle can then be reacted with a FA molecule conjugated to avidin or streptavidin to form a biotin-avidin or biotin-streptavidin complex.

For example, in one embodiment, an amino-functionalized Pt nanoparticle (where an amino group is accessible and not bound to the Pt surface) can be reacted with an amine-reactive biotinylation reagent to functionalize the nanoparticle with biotin. Some examples of amine- reactive biotinylation reagents include the class of molecules containing biotin on one end and, for example, a succinimide ester, pentafluorophenyl ester, or alkyl halidc group on the other end. The biotin group and amine-reactive group can be separated by any suitable spacer group of any length (e.g., 8-40 A in length). Some examples of amine-reactive biotinylation reagents are available from Pierce under the EZ-Link© trade name, e.g., as NHS-biotin (containing a five- carbon ester linkage between biotin and NHS), sulfo-NHS-biotin, NHS-LC-biotin, and sulfo- NHS-LC-Biotin, NHS-LC-LC-biotin, sulfo-NHS-LC-LC-biotin, sulfo-NHS-SS-biotin, NHS- PE0₄-biotin, PFP-biotin, TFP-PEO-biotin, and the like, wherein "NHS" refers to a N- hydroxysuccinimide group, "LC" refers to a six-carbon amide-containing linkage inserted between the NHS group and biotin or between another LC group and biotin, "PEO" refers to an ethyleneoxide group, wherein the associated subscript indicates the number of linked PEO units, "PFP" refers to a pentafluorophenyl group, "TFP" refers to a tetrafiuorophenyl group, "sulfo" refers to a sulfonate (S02 Na⁺) group, and "SS" refers to a disulfide bond.

In another embodiment, a thiol-functionalized Pt nanoparticle (where a thiol group is accessible and not bound to the Pt surface) can be reacted with a thiol-reactive biotinylation reagent to functionalize the nanoparticle with biotin. Some examples of thiol-reactive biotinylation reagents include the class of molecules containing biotin on one end and, for example, a maleimido or alkyl halide group on the other end. The biotin group and thiol-reactive group can be separated by any suitable spacer group of any length, as above. Some examples of thiol-reactive biotinylation reagents are available from Pierce under the EZ-Link® trade name, e.g., as maleimide-PEOlbiotin. biotin-BMCC (contains an end-maleimido group and one cyclohexyl, two amide linkages, and nine additional linking carbon atoms), PEO-iodoacetyl biotin, iodoacetyl-LC-biotin, biotin-HPDP (contains a pyridyl disulfide group), and the like. The biotin-functionalized Pt nanoparticle can then be reacted with an avidin or streptavidin conjugate of a FA molecule such that a FA-Pt nanoparticle complex is produced which contains the nanoparticle conjugated to the FA molecule by a biotin-avidin or biotin- streptavidin link.

In an analogous embodiment, the platinum nanoparticle is conjugated to avidin or streptavidin and the avidin- or streptavidin- functionalized nanoparticle reacted with a biotinylated FA molecule such that a FA-Pt nanoparticle complex is produced which contains the nanoparticle conjugated to the FA molecule by an avidin-biotin or streptavidin-biotin link.

The FA-Pt complex can also include groups that can make the complex visible by one or more diagnostic techniques. Some observable groups include, for example, paramagnetic and radionuclide species conjugated to the FA-Pt complex of the invention. Some examples of diagnostic techniques include magnetic resonance imaging (i.e., MRI) and positron emission detection techniques (e.g., Positron Emission Tomography (PET) or Single Photon Emission Computed Tomography (SPECT)).

Since the FA-Pt nanoparticle complexes described above are to be administered to a subject, the probes can be optionally combined with a suitable pharmaceutical carrier (i.e., vehicle or excipient). Any of the excipients known in the art can be suitable herein depending on the mode of administration. Some examples of suitable carriers include gelatin, fatty acids (e.g., stearic acid) and salts thereof, talc, vegetable fats or oils, gums and glycols, starches, dextrans, and the like. A pharmaceutical composition of the FA-Pt nanoparticle complex can also include one or more stabilizers, surfactants, salts, buffering agents, additives, or a combination thereof. The stabilizer can be, for example, an oligosaccharide (e.g., sucrose, trehalose, lactose, or a dextran), a sugar alcohol (e.g., mannitol), or a combination thereof. The surfactant can be any suitable surfactant including, for example, those containing polyalkylene oxide units (e.g., Tween 20, Tween 80, Pluronic F-68), which are typically included in amounts of from about 0.001% (vv/v) to about 10% (w/v). The salt or buffering agent can be any suitable salt or buffering agent, such as, for example, sodium chloride, or sodium or potassium phosphate, respectively. Some examples of additives include, for example, glycerol, benzyl alcohol, and 1 ,1 ,1 -trichloro-2- methyl-2-propanol (e.g., chloretone or chlorobutanol). If required, the pH of the solutions can be suitably adjusted and buffered.

In another aspect, the invention is directed to a method for the treatment of cancer or a pre-cancerous condition (i.e., neoplastic condition) in a mammal by administering the above- described composition to the mammal in a treatment-effective (i.e., effective) amount. The mammal can be any mammal (e.g., a cat, dog, horse, or ape), but the method is more typically directed to human subjects.

The cancer or pre-cancerous condition (neoplastic condition) can be located in any internal organ of the body. Some examples of applicable body parts containing cancer cells include the heart, lungs, stomach, intestines, breasts, prostate, ovaries, pancreas, kidney, liver, bladder, uterus, colon, or rectum. The cancer or neoplasm can also include the presence of one or more carcinomas, sarcomas, lymphomas, blastomas, or teratomas (germ cell tumors). The cancer can also be a form of leukemia. The platinum-folate composition specifically (i.e., selectively) binds to (i.e., targets) neoplastic cells while exhibiting a substantial absence of binding to non-cancerous cells. By selectively binding to neoplastic cells, the composition selectively targets and destroys neoplastic cells (i.e., preferentially induces apoptosis in cancerous or pre-cancerous cells) while not damaging normal cells. The method for targeting neoplastic cells in a subject involves administration of the above-described FA-Pt nanoparticle complex (i.e., FA-Pt complex) into a subject. The FA-Pt complex can be administered to the subject in any suitable manner that can allow the probe to selectively target neoplastic cells. For example, the FA-Pt complex can be administered externally (i.e., orally), parentally (i.e., by infusion through the skin), topically (i.e., on the skin), or by injection (e.g., intravenously or intramuscularly). For oral administration, liquid or solid oral formulations can be given. These include, for example, tablets, capsules, pills, troches, elixirs, suspensions, and syrups.

The FA-Pt complex is administered in a treatment-effective amount. A treatment- effective amount is an amount which causes the selective apoptosis of neoplastic cells. The amount of FA-Pt complex administered depends on several factors, including the solubility and size of the FA-Pt complex, weight of the subject, and other factors. A treatment-effective amount is typically within the range of, for example, 10-1000 mg of the complex per administration. In different embodiments, the complex is administered at or above, or at or below 5 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1200 mg, or 1500 mg per administration, wherein the complex can be administered by any suitable schedule, e.g., once daily, once weekly, twice daily, or twice weekly. The FA-Pt complex can also be administered in a way which releases the complex into the subject in a controlled manner over time (i.e., as a controlled release formulation). The complex can also be administered along with other cancer drugs, adjuvants, or auxiliary agents commonly known in the art.

In a first preferred embodiment, the FA-Pt complex exhibits a higher cellular uptake in neoplastic cells as compared to the uptake in normal cells. Accordingly, the ratio of cellular uptake of platinum nanoparticlcs in neoplastic cells compared to normal cells (i.e., the "cellular uptake ratio") is preferably greater than 1 percent. In different embodiments, the cellular uptake ratio is preferably at least about or greater than 1.5, or 2.0, or 2.5, or 3.0, or 3.5, or 4.0 percent. Preferably, the cellular uptake of platinum nanoparticles in neoplastic cells is at least about 20 percent, and more preferably, at least about 25, 30, or 35 percent. Preferably, the cellular uptake of platinum nanoparticles in normal cells is no more than about 15 percent, and more preferably, no more than about 12.5, 10,9, 8,7,6, 5, or 4 percent.

In a second preferred embodiment, an effective amount of the FA-Pt complex is an amount which corresponds to an IC50 fold value of at least about or greater than 1.0. More preferably, an effective amount of the FA-Pt complex is an amount which corresponds to an IC50 fold value of at least about or greater than 1.25, and even more preferably, an IC50 fold value of at least about or greater than 1.5, 1.75, 2.0,2.25, or 2.5. Alternatively, an effective amount of the FA-Pt complex is an amount which corresponds to an apoptosis fold value of at least about or greater than 1.0. More preferably, an effective amount of the FA-Pt complex is an amount which corresponds to an apoptosis fold value of at least about or greater than 1.5, and more preferably, higher apoptosis fold values of at least about or greater than 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0.

As used herein, the term 'IC₅₀' is used to represent the half maximal (50%) inhibitory concentration of the FA-Pt nanoparticle complex. More specifically, the IC50 value as defined herein refers to the concentration of nanoparticles required to inhibit the growth of one-half of the cells after 24 hours from the time treatment of the cells with the complex has started.

As used herein, 'ICjo fold' means a concentration that is a multiple or fraction of an IC50 value. Thus, for example, a two-fold IC₅₀ value (i.e., an IC50 fold of 2) indicates twice the IC50 concentration. As used herein, 'apoptosis fold' means the proportion of cells that are apoptotic in a sample of cells treated with the nanoparticle complex, as compared to the number of cells that are apoptotic in a sample that is untreated (control). For example, if 8% of the cells are apoptotic in a sample of cells treated with the nanoparticle complex, and 4% of the cells are apoptotic in a sample that is untreated, this represents a two-fold increase in the proportion of apoptotic cells in response to treatment, and thus, an apoptosis fold of 2.

Examples have been set forth below for the purpose of illustrating and describing this preferred embodiment of the invention.

EXAMPLE 1 : Preparation of a FA-Pt Nanoparticle Complex

The FA-coated Pt nanoparticles were synthesized as follows. An Erlenmeyer flask was charged with 0.005 mol of potassium tetrachloroplatinate(II) (K₂PtCl₄) and 0.005 mol of FA (C 1911 HQN_VO₆) in 20 ml of deionized water. The Erlenmeyer flask containing this solution was then heated on a hot plate to approximately 86⁰C for about 20 minutes to fully dissolve the potassium tetrachloroplatinate and FA. After allowing the solution to cool down for about 10 minutes at room temperature, 0.1 ml of 0.0189 M sodium borohydride (NaBH₄) was slowly added to the cooled solution. Immediately after the reductant was added, the color of the solution changed from orange to black, thus indicating production of Pt nanoparticles (Pt-NPs).

According to the TEM image shown in Fig. 15 and the size distribution graph of Fig. 16, the Pt nanoparticles have an average size of about 4.5 nm, with a standard deviation of about 0.25 nm. The TEM image is under 40 nm scale.

EXAMPLE 2: Cell Lines Two different cell lines were used in this study. MCF 7 (mammary breast cancer cells) and MCF 1OA (mammary breast cells) were ordered from ATCC. MCF7 cells were obtained from a 69 year-old Caucasian female. MCF 1OA cells were obtained from a 36 year-old Caucasian female. MCF7 cells were cultured under ATCC-formulated Eagles' Minimum Essential Medium with O.Ol mg/ml bovine insulin and fetal bovine serum to a final concentration of 10%. MCF 1 OA cells were cultured under Lonza single kit medium with 2.5 niM L-glutamine and supplemented with 20 ng/ml epidermal growth factor, 100 ng/ml cholera toxin, 0.01 mg/ml insulin and 500 ng/ml hydrocortisone, 95% and 5% horse serum. Both cells were incubated at 37 ⁰C with 5% CO₂.

EXAMPLE 3: MTT Experiment and Results A tetrazolium salt (MTT) (3-{4,5-dimethylthiazol-2-yU-2,5-diphcnyltetrazolium bromide) labeling reagent in vitro toxicology assay kit supplied by Sigma (Cat No. M 5655 and M 8910) was used to define Pt-NP toxicity on both cell lines of MCF7 and MCF 1 OA. Both cells were seeded in 96-well plates with cell density of 1.4 x 10⁴ and 100 μl medium in each well. After cells were seeded overnight, the medium was removed under a laminar flow hood.

The first column of wells was left blank, which was used to subtract the background absorption of the 96-well plate. A control was placed in the second and the third columns. In the second column, only 100 μl complete medium (please see Example 2, i.e. the complete medium is Minimum Essential Medium, Eagle, (catalogue number is 30-2003, which was ordered from ATCC)), together with 10% fetal bovine serum. The complete medium was pipetted into each well. In the third column, synthesis solution without K₂PtCU was pipetted into each well. The previous medium was completely removed. Pt-NPs were dissolved in the complete medium in a scries of concentrations, and this followed by pipetting 100 μl of the Pt-NP/complete medium solution into each well. 96-well plates were incubated for another 24 hours at 37°C under 5% CO₂. After 24 hours of incubation, 10 ml of reconstituted MTT was introduced to each well, respectively (no blank wells). The plates were gently moved to let reconstituted MTT mix uniformly in each well. After another 3 hours of incubation, the resulting formazan crystals were dissolved by adding 100 μl MTT solubilization solution (cat no: M 8910) by pipetting up and down in order to completely dissolve the MTT formazan crystals. Absorbance was measured by a SpectraMax method at a wavelength of 570 nm. MTT results are shown in Tables 3 and 4 below, as detected by SpectraMax. Table 3 summarizes results of an MTT study on Pt-NPs with MCF7 cells and Table 4 shows IC₅₀ results on Pt-NPs with MCF7 and MCF 1 OA cells.

Table 3

1 2 3 4 5 6 7 8 9 10 1 1 12

A

B Blank Ctrl 1 Ctrl 2 5.0 10.0 15.0 20.0 25.0 30.0 35.0

C Blank Ctrl 1 Ctrl 2 5.0 10.0 15.0 20.0 25.0 30.0 35.0

D Blank Ctrl 1 Ctrl 2 5.0 10.0 15.0 20.0 25.0 30.0 35.0

E Blank Ctrl 1 Ctrl 2 40.0 45.0 50.0 55.0 — —

F Blank Ctrl l Ctrl 2 40.0 45.0 50.0 55.0 — —

G44 Blank Ctrl 1 Ctrl 2 40.0 45.0 50.0 55.0 — —

H Cone, unit is μg/ml. Ctrll is MCF7 with complete medium. Ctrl2 is Pt-NPs synthesis solutions without K₂PtCl₄

Table 4 is an MTT study on Pt-NPs with MCFlOA cells. Table 4

I 2 3 4 5 6 7 8 9 10 U 12

A

Blan Ctrl Ctrl 5.0 10.0 15.0 20.0 25.0 30.0 35.0

B k 1 2

Blαn Ctrl Ctrl

C 5.0 10.0 15.0 20.0 25.0 30.0 35.0 k 1 2

Blan Ctrl Ctrl

D 5.0 10.0 15.0 20.0 25.0 30.0 35.0 k I 2

Blαn Ctrl

E Ctrl 55.0 — ...

40.0 45.0 50.0 k I 2

Blan Ctrl

F Ctrl

40.0 45.0 50.0 55.0 ... k I 2

Ctrl

G44 blank Ctrl 40.0 45.0 50.0 55.0

I 2

H

Cone, unit is mg/ml

Ctrl l is MCFIO with complete medium

Ctrl2 is Pt-NPs synthesis solutions without KiPtCU

Table 5 provides IC₅₀ results on Pt-NPs with MCF7 and MCFlOA cells. Table 5

The MTT graphs shown in Figs. 17 and 18 were obtained from SpectraMax. The x-axis indicates the concentration of nanoparticles and the y-axis indicates cell growth rate. The graphs are used to find IC₅₀ values (i.e., the concentration of the complex at 50% cell growth rate). Fig. 17 shows the MTT result, which shows that the concentration of Pt-folate NPs necessary to induce 50% cell growth inhibition (i.e., allow for 50% overall cell growth) in neoplastic cells is 25 μg/mL. In contrast, Fig. 18 shows that the concentration of Pt-folate NPs necessary to induce 50% cell growth inhibition (i.e., allow for 50% overall cell growth) in normal cells is approximately 29 mg/niL (-29,000 μg/mL).

MTT results in comparison to MCF 7 cells and MCF 1 OA cells are listed in Table 3, respectively, which shows great selectivity of Pt-NPs on both cell lines. It is evident that a small amount of Pt-NPs can greatly affect MCF7 cell growth, while MCF 1 OA cells exhibit 50% cytotoxicity under much higher concentration of Pt-NPs. EXAMPLE 4: Uptake Study Experiments and Results

Both MCF7 and MCF 1 OA cells were seeded in a 96-well plate with a cell density of 1.4 x 10⁴ cells/well overnight. Pt nanoparticles were added as 10 μil to each well for another 24 hrs incubation at 37 ⁰C and under 5% CO₂. Control cells were cells without introducing any Pt nanoparticles. After incubation, the supernatant was carefully removed and each well was washed with PBS twice to clean up residual Pt nanoparticles which were not absorbed by the cells. Pt-NPs remaining in the medium were carefully collected by centrifuge and allowed to dry, before being weighed on a microbalance. Trypsinized cells together with supernatant (which was previously removed) was centrifuged at 1200 rpm for 5 min. Supernatant was then removed. Pelleted cells were then analyzed using a TGA/SDTA 85 l^c thermogravi metric analyzer to elucidate their cellular uptakes. Three different controls were used: MCF7 cells, MCFlOA cells and pure Pt-NPs solution.

Table 6, of Pt-NPs cellular uptake studies by TGA/ SDTA 85 l^c, shows that Pt nanoparticles have more uptake in MCF 7 (35.73% ± 4.14%) than in MCF 1OA cells (9.43% ± 2.24%), showing that Pt nanoparticles have good selectivity between both cell lines. Table 6

Average Standard deviation

Control-MCF7 /mg 0.00003 0.00001 0.00005 0.00003 ±1.63e-5

Control-MCFIOA/mg 0.00005 0.00002 0.00004 0.00004 ±1.26e-5

Control-Pt-NPs 1.04 0.91 0.94 0.96 ±0.06

Pt-NPs left in 0.55 0.59 0.50 0.55 ±0.04 medium/mg

MCF7 Pt-NPs in cells/mg 0.27 0.30 0.34 0.28 ±0.04

Overall Pt- 0.82 0.89 0.84 0.85 ±0.03 NPs/mg

Pt-NPs 33.0 33.7 40.5 35.73 ±4.14 uptake/%

Pt-NPs left in 0.77 0.74 0.64 0.72 ±0.06 medium/mg

MCF Pt-NPs in

0.1 1 0.07 0.05 0.08 ±0.03 cells/mg

1OA Overall Pt-

0.88 0.81 0.69 0.79 ±0.08

NPs/mg

Pt-NPs 12.50 8.60 7.20 9.43 ±2.24 uptake/%

EXAMPLE 5: Apoptosis Study Experiments and Results

The apoptosis study was performed using the Cell death detection Elisa plus kit supplied by Roche (cat no. 1 1774425001 ) and following the instructions provided by the manufacturer. Each cell line was seeded in a 96-well plate at a density of 1.4 x 10⁴ cells and after 24 hours of incubation at 37 ⁰C and 5% CO₂, the cell pellet was re-suspended in 200 μl Lysis Buffer (kit provided), and incubated for 30 minutes at 20 ⁰C. The lysate was centrifuged at 200 x g for 10 minutes. 20 μl culture supematants after centrifuge and treatment (CAM), 20 μl lysates of CAM treated cells after centrifuge, 20 μl positive control (kit provided), 20 μl negative control (culture supernatant and lysate after centrifugation of untreated cells) and 20 μl background control (incubation buffer, kit provided) were carefully transferred into the streptavidin-coated microplate for analysis. 80 μl of the immunoreagcnt was added to each well and the microplate covered with foil paper. The microplate was then incubated on a microplate shaker at 300 rpm gentle shaking for 2 hours at 20 °C. The solution was removed thoroughly after 2 hours and each well was rinsed with 300 μl Incubation Buffer (kit provided) three times. The solution was carefully removed each time. The 100 μL ABTS (kit provided) was pipetted to each well, following 100 μl ABTS Stop Solution (kit provided). The results were obtained by measuring the absorption at 405 nm against ABTS solution and 100 μl ABTS Stop Solution as a blank using the SpectraMax multi-detection microplate reader. Referring to Fig. 19, it is shown that at lower IC₅₀ fold both cancer and normal cells behave similar to their controls. However, as shown, with increasing IC₅₀ fold value the apoptosis fold amount of breast cancer cells dramatically increases. Furthermore, little change is shown with IC50 fold variations. The above results demonstrate that Pt-NPs can greatly induce breast cancer cell apoptosis especially at higher IC₅₀ fold value. Moreover, the FA-Pt nanoparticle studied herein shows little effect on induction of normal breast cell apoptosis within the same IC50 fold range. These results demonstrate that the folate-platinum nanoparticles described herein selectively target and preferentially induce apoptosis of cancerous cells over normal cells.

EXAMPLE 6: Dihydroethidium Fluorescence Experiments and Results Both MCF 7 and MCF 1 OA cells were seeded in a 96-well plate at a cell density of 1.4 x

10⁴ cells/well overnight. 10 μl of 5 μM dihydroethidium (DHE) was added to each well, keeping it at 37 ⁰C and 5% CO₂ for 30 min. Pt-NPs were incubated for 1 hour, 2 hours, 3 hours and 4 hours. Cells without Pt-NPs were kept as controls. Absorbance was measured at an excitation of 518 nm and an emission of 605 nm by a SpectraMax M5 multi-detection microplate reader. Both MCF 7 and MCF 1 OA cells were also studied by flow cytometry. Cells were seeded in 6-well plate at a cell density of 0.4 x 10^(> cells/well overnight, followed by three hours Pt-NPs incubation. A control was conducted by using cells without Pt-NPs. Cells were collected after twice PBS washing and centrifuge. 500 μl of 5 μM DHE was added to each well and incubated for 30 minutes. A column of cells were not dyed by DHE due to background reference consideration.

Figs. 20-22 show the results of the detection of Reactive Oxygen Species (ROS) from the cell lines by using either SpectraMax or flow cytometry. ROS include a variety of including, for example, superoxide anion, hydrogen peroxide, singlet oxygen, peroxynitrite anion, hydroxyl radical, and nitric oxide. These ROS molecules are normally produced at detectable levels during apoptosis. These species can be detected by dihydroethidium (DHE), a dye which is the dihydrogenated version of ethidium. This dye is oxidized by superoxide, resulting in the release of ethidium which fluoresces when bound to cellular DNA.

The results shown in Figs. 20-22 demonstrate that the folate-coated Pt nanoparticles of the invention cause breast cancer cells to produce superoxide in far greater amounts than normal breast cells, and thus, evidences the preferential induction of apoptosis in breast cancer cells over normal breast cells. While there have been shown and described what are presently believed to be the preferred embodiments of the present invention, those skilled in the art will realize that other and further embodiments can be made without departing from the spirit and scope of the invention described in this application, and this application includes all such modifications that are within the intended scope of the claims set forth herein.

Claims

WHAT IS CLAIMED IS;

1. A biosensor comprising at least one biosensing monolayer, wherein said monolayer comprises a gold coated substrate and alkanethiol molecules with hydroxyl end groups chemically bound thereto, wherein at least one target molecule is present during formation of said monolayer, followed by removal of said target molecule from said monolayer thereby forming an imprint of the target molecule for which detection is sought on said monolayer.

2. The biosensor of claim 1 , wherein the at least one target molecule comprises a protein fraction from a bacteria, virus, or target protein.

3. The biosensor of claim 1 , wherein the at least one target molecule is a cancer biomarker.

4. The biosensor of claim 1, wherein the at least one target molecule is carcinoembryonic antigen.

5. The biosensor of claim 1 , wherein the at least one target molecule is fibrinogen, hemoglobin, pseudomonas aeruginosa, staphylococcus aureus, fibronectin, fibrinogen, osteocalcin, or combinations thereof.

6. The biosensor of claim 1, further comprising an array on two or more monolayers.

7. The biosensor of claim 1 , wherein the alkanethiol molecules form a molecular monolayer atop the gold via sulfur-metal bonds.

8. The biosensor of claim 7, wherein the at least one target molecule is embedded in the alkanethiol monolayer via hydrophilic interactions with hydroxyl groups of the alkanethiol molecules.