Today malignant tumors, especially liver cancer, are the primary cause of the death in Taiwanese citizens. According to research reports, moderate liquor consumption helps maintain human health; however, consuming excessive amounts of alcohol leads to diseases such as liver cancer and acute alcoholism. In our research, we discovered that alcohol dehydrogenase metabolizes alcohol into acetaldehyde, which is then converted by aldehyde dehydrogenase into acetic acid in the body. Acetic acid is dramatically more biologically compatible with the human body than ethanol. In our project, we modified the surface of the magnetic nanoparticle Fe3O4 to provide a platform to which enzymes could attach and to allow us to analyze the properties of the combined particle containing both enzyme and Fe3O4 nanoparticle. The first experimental enzyme we chose was Trypsin; as our research techniques matured, we used alcohol dehydrogenase and aldehyde dehydrogenase to conduct our research. Finally, we used instruments to compare the results to understand the effectiveness of both the surface modification and the enzyme attachment, in order to judge our ultimate goal - whether or not the experiment was successful.

Traditional medication treatment is a process in which medicine enters human body through injection or orally absorption. Traditional medication treatment does not own the property of target aiming; moreover, it often lowers the effectiveness of the medicine, hence enhances the possibilities of patients‘ side effects. However, the time of treatment and the dosage of medicines will be reduced when medicines were modified on Fe3O4 nanoparticles and carried to targeted object by magnetic fields. In our research, we utilize magnetic Fe3O4 nanoparticles as carriers of enzymes - Alcohol dehydrogenase (ADH) and Aldehyde dehydrogenase (ALDH) to reach the ultimate goal effectively: metabolizing the more baleful alcohol into acetic acid in human bodies.

Alcohol    (Ethanol) Acetaldehyde Acetic  Acid

Alcohol  Dehydrogenase  (ADH)  

aldehyde  dehydrogenase      (ALDH)  

Medicine 1 Ferrous chloride tetrahydrate 2 Iron (III) chloride hexahydrate 3 Hydrochloric Acid 4 Sodium hydroxide 5 L-DOPA 6 EDC 7 NHS 8 L-BAPNA 9 Trypsin Type XII-S

10 Dimethyl Sulfoxide 11 PBS (pH=7.4) 12 Alcohol dehydrogenase (ADH) 13 Aldehyde dehydrogenase (ALDH) 14 Ethanol 15 Water 16 NAD+

Instruments 1 UV-Vis Absorption Spectrometer 2 Atomic Absorption Spectrometer (AA) 3 Transmission Electron Microscopy (TEM) 4 X-ray Diffractometer (XRD) 5 Fourier Tranform Infrared (FT-IR) 6 Dynamic Light Scattering (DLS) 7 Enzyme-Linked ImmunoSorbent Assay reader (ELISA reader) 8 pH meter 9 Sigma Scan Pro 5

Make magnetic Fe3O4 nanoparticles

Surface modification (-NH2) of nanoparticle

Examination by instrument AA to do quantitative analysis

Examine by TEM, XRD, Zeta-potential and FT-

IR

Connect experimental enzyme

- Trypsin

Connect final enzyme - alcohol dehydrogenase

Connect final enzyme - aldehyde

dehydrogenase

Use UV-vis and pH meter to determine if alcohol is metabolized into acetic acid

Determine the result by substrate - BAPNA

Discussion of research and data analyses

Biomedical Applications of Magnetic Fe3O4 Nanoparticles

1. Use chemical co-precipitation to make magnetic Fe3O4 nanoparticles 2Fe3+ + Fe2+ +8OH- Fe3O4 + 4 H2O 2. Mix Fe3O4 particles with L-DOPA in the molecule ratio of 1:10000; the precipitation of the mixture is Fe3O4~NH2

Preparation of amino-magnetic Fe3O4 nanoparticles (Fe3O4~NH2)

Fe3O4 Fe3O4~NH2

RESULTS

FT-­‐IR  analyses  of  Fe3O4  and  Fe3O4~NH2 XRD  analyses  

Connection of enzyme Trypsin and Fe3O4~NH2 1.  Mix 1 mL magnetic Fe3O4 nanoparticles(aq), 0.5mM Trypsin 190.5µL, 10mM EDC 9.5µL and 10mM NHS 9.5µL. (A mixture with the

nanoparticles and enzymes ratio of 1:250) 2.  Use ELISA reader to analyze mixture with BAPNA and without BAPNA. Record data every 10 minutes.

RESULTS

Connection of enzyme ADH/ALDH and Ethanol

1. Mix ADH, Fe3O4~NH2, EDC and NHS, stir the solution for 2 hours 2. Mix ALDH, Fe3O4~NH2, EDC and NHS, stir the solution for 2 hours

RESULTS Enzyme Avg Size (nm)ADH 24.751

ALDH 34.698Trypsin 35.45

Abs in 267 nmstandardized solution 1.74636

clear supernatant liquid 0.76927standardized solution 1.14702

clear supernatant liquid 0.5015

Materials

ADH

ALDH

Known  from  the  table  (the  ideal  combination  is  250  enzymes  /  nanoparticle)   ADH:  139.88  (enzymes  /  nanoparticle) ALDH:  140.69  (enzymes  /  nanoparticle)

L-DOPA has been successfully connected to Fe3O4 particle, XRD shows that the magnetic property remains in this experiment

Shows that if given enough time, enzyme and the substrate will completely function. It also shows that enzyme can function with Fe3O4~NH2

Use UV-vis to analyze the absorption spectrum of material A, B, C A: Fe3O4 nanoparticles with ADH, ALDH + NADH + 50% alcohol: collect data immediately after alcohol was added B: Fe3O4 nanoparticles with ADH + NADH + 50% alcohol C: Fe3O4 nanoparticles with ADH, ALDH + NADH + 50% alcohol: collect data 10 minutes after alcohol was added

Biomedical Applications of Magnetic Fe3O4 Nanoparticles

A. Making magnetic nanoparticles Common methods of making magnetic nanoparticles include grinding method, chemical co-precipitation method...and so on. However, chemical co-precipitation method is the most widely used method to make magnetic nanoparticles. Moreover, chemical co-precipitation contains other properties such as low cost, simple operation, short time, and could be massively produced. Hence, we choose chemical co-precipitation to make nanoparticles in our experiment. B. The chelation of surface modification In this experiment, it is the amino function group (~NH2) of L-DOPA that provides the derivative function to the nanoparticle through surface modification. L-DOPA connects to a surface of the magnetic nanoparticles by chelation. Chelation is a way to combine metal with either ion or molecules. It often occurs in a ligand, which is normally an organic compound, also known as the chelating agents. L-DOPA is an excellent chelating agent; it chelates and combines with iron, connecting onto the surface and providing attachment places for the enzyme. C. The selection of enzymes In the primary stage of the experiment, we choose Trypsin (a type of pancreas enzyme) as the enzyme to connect. Normally, Trypsin functions in small intestines and hydrolyzes proteins to amino acids, which can be absorbed by human bodies. However, we select alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) in our final experiment. Due to the high prices of ADH and ALDH, we use Trypsin as an experimental enzyme to practice our experimenting skills and do quantitative analysis. After our experimenting techniques and process were matured, we use ADH and ALDH to conduct finalized data analyses. D. The ratio of enzymes and magnetic nanoparticles In this experiment, we set up the ideal ratio of enzymes per nanoparticle is 1:250. In our previous experiments, we found out that 1:250 has the best connection result among 1:100, 1:250 and 1:500. In addition to the experiment results, we hope that nanoparticles connect with suitable amount of enzymes so that we could reach the best efficiency while data analyzing through instruments. Therefore, we considered that one nanoparticle with 250 enzymes is the best ideal ratio in this experiment. Also, other research papers recorded that a nanoparticle with a radius of 3 nm contains 1785 Fe3O4 particle numbers. Hence, we get the Fe3O4 particle numbers by TEM results and thus figured out the manufacturing of 1/250 ratio. E. The effect of EDC and NHS EDC and NHS will activates the COOH functional group. First EDC will function with -COOH and form R1—COO—EDC. Then NHS will react with this group and form R1—CO—NHS. Afterwards, R1—CO—NHS will react with R2—NH2 and form the compound R1—C=O—NH—R2, which is stable in chemical reactions. F. The absorption method of enzymes 1.  Absorbed though diet: (Advantages) convenient. (Disadvantages) enzymes may be damaged through the violent variation of pH in human bodies 2. Absorbed through the injection of magnetic nanoparticles: (Advantages) extend the activity of enzymes, maintain the stability of nanoparticles, can control the path of transportation of enzyme. G. The selection of instruments in analyzing the metabolism of alcohol   In the data analyzing of alcohol, the ideal instrument was HPLC with 18C column. HPLC utilizes the different properties of material absorptions to the column in order to separate alcohol, aldehyde and acetic acid. However, because HPLC's column is made up of organic materials, which is the same as alcohol, aldehyde and acetic acid, these three substances will barely have differences in separation. This makes data analyzing difficult because the signals will overlap with each other. As a result, we use pH meter to detect the result because of the acid property of acetic acid.

1.  From the results of Zeta Potential, Fe3O4 has negative charge but Fe3O4~NH2 has positive charge, which demonstrates the success of surface modification. Moreover, because of the positive charges in Fe3O4~NH2 , the charges will reject each other, which offers the nanoparticles to spread evenly in the water. This effect decreases the effect of blockage in circulatory system.

2.  From the results of Zeta Potential, Nanoparticles will change their surface potentials while connected to Trypsin, ADH and ALDH. 3.  From the analyses of FT-IR, we observed signals of benzene on Fe3O4 nanoparticles. Therefore, it is evidenced that L-DOPA is attached

to the surface of nanoparticles. 4.  From the XRD results, we could observed the fact that Fe3O4 nanoparticles still keep the magnetic characteristics after attaching L-

DOPA and enzymes. 5.  From TEM photographing, Fe3O4~NH2 with Trypsin has a larger particle size than ones without Trypsin. 6.  From ELISA reader analyses, we observed that if given enough time, different concentration of enzyme (Trypsin) can fully interact with

substrate (BAPNA) 7.  From UV-vis results, the number of enzymes attached to a nanoparticle is 56% of the ideal amount. 8.  From the results of UV-vis, alcohol (before reaction), aldehyde (after adding ADH) and acetic acid (after adding ADH and ALDH) have

different crest in the absorption spectrum, which we believed that it is the C=O bond that causes the difference. In this analysis, we know that enzymes did metabolize alcohol into acetic acid.