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W tym Artykule

  • Podsumowanie
  • Streszczenie
  • Wprowadzenie
  • Protokół
  • Wyniki
  • Dyskusje
  • Ujawnienia
  • Podziękowania
  • Materiały
  • Odniesienia
  • Przedruki i uprawnienia

Podsumowanie

The synthesis of asymmetric species of ferrocene is challenging using solution techniques. This report focuses on the methods carried out to produce a ferrocene-biotin bioconjugate using facile and clean reactions accomplished via solid-phase synthesis. Incorporation of a thiolate moiety is shown to impart the ability for immobilization on gold surfaces.

Streszczenie

Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. The most common techniques utilized are imaging modalities such as Magnetic Resonance Imaging (MRI), Positron Emission Topography (PET), and Computed Topography (CT) and are optimal for understanding the physical structure of the disease but can only be performed once every four to six weeks due to the use of imaging agents and overall cost. With this in mind, the development of “point of care” techniques, such as biosensors, which evaluate the stage of disease and/or efficacy of treatment in the clinician’s office and do so in a timely manner, would revolutionize treatment protocols.1 As a means to exploring ferrocene based biosensors for the detection of biologically relevant molecules2, methods were developed to produce ferrocene-biotin bio-conjugates described herein. This report will focus on a biotin-ferrocene-cysteine system that can be immobilized on a gold surface.

Wprowadzenie

Biosensors are small devices that employ biomolecular recognition technology as the platform for selective analysis and are utilized for their specificity, speed, and low-cost. Electrochemical biosensors for the detection of biomolecules are at the forefront of this field due to their simplicity, cost effectiveness, and high sensitivity.1,3 The general anatomy of these sensors is an electrode equipped with a recognition molecule specific for the biological marker of interest. Binding of the biomarker by the recognition molecule results in a local change of potential or current that can be detected by simple measurement. To date the recognition moiety can range from enzymes,4-8 antibodies,9-12 whole cells,13-16 receptors,17-20 peptides21-23 and DNA24 and have largely focused on larger, biological molecules.25-28 Research efforts in this arena have concentrated mainly on immunosensors where an immunoglobulin is immobilized with a redox active core (such as ferrocene) and used to detect an antibody of interest. These studies have been excluded from clinical applications due to poor precision and time consumption stemming from the complications arising from use of antigen/antibodies.1,3 Growing attention has focused on the detection of small molecules (less than 1 kg/mol) of biomedical, food and environmental interest in addition to national security.29 The best known examples of biosensor devices are self-test glucose monitors, which have screen printed enzyme electrodes coupled to a pocket-size amperometric meter. These systems typically utilize a coulometric method where the total amount of charge generated by the glucose oxidation reaction is measured over a period of time. Marketable devices must be portable, robust and hand-held to make use facile for the population at large.

Redox tags such as ferrocene are necessary to provide the electrochemical detection of biomarkers or small molecules in solution as most biomarkers are not intrinsically electrochemically active.30-38 Ferrocene is an organometallic molecule that is a gold standard for electrochemistry, which makes it an excellent choice for integration into electrochemical biosensors. Ferrocene-based redox active species have already garnered considerable attention due to their small size, good stability, convenient synthetic access, easy chemical modification, relative lipophilicity, and ease of redox tuning.3,30-42 Small molecules based on the ferrocene core have been used extensively as detectors of metal ions and small molecules.32-38,43 Systems targeting larger species such as biomolecules have utilized the attachment of large antibodies or immunoglobulins to ferrocene derivatives that have been embedded onto an electrochemical surface.1,3,39,44 In each case, the potential and current intensity of the FeIII/FeII redox couple was altered upon molecular coupling, thus producing a new spectroscopic handle indicating the presence of the analyte molecule. This change arises from the extensive overlap that occurs between the pi-system of the cyclopentadienyl rings and the iron d-orbitals. If the pi-system is modified, i.e., derivatized or reacted, then the orbital interaction will, in turn, change. This will affect the Fe core and can be observed as a shift in the potential of the FeIII/FeII couple.40,45,46 These properties make such a system attractive for use as a quantifying agent in an electrochemical immunoassay or biosensor.

In order to produce ferrocene containing systems specific for biosensor capacities it is optimal to modify one Cp ring with the bio-receptor specific for a target molecule and utilize the other Cp ring as a molecular tether to the electrochemical readout or electrode (Figure 1). Synthesis of these asymmetric ferrocene derivatives is challenged by side reactions and the formation of dimeric and polymeric species formed upon intermolecular cross-linking.47 However, coupling chemistry producing an amide bond is the most direct route to provide simple derivatives of ferrocene involving biological components such as peptides and their metabolites. Therefore, solid phase techniques first developed in the 1950s by Merrifield for peptide synthesis can be applied to organometallic compounds containing ferrocene. Through the use of the orthogonally substituted 1’-Fmoc-amino-ferrocene-1-carboxylic acid molecule, a ferrocene system that can contain a receptor moiety (biotin), electrochemical readout (ferrocene), and immobilizing-linker component (cysteine) has been constructed and detailed herein. The synthesis of this bio-conjugate is discussed as well as evidence for immobilization on a gold surface. This work represents the first presentation of a system composed of biotin, ferrocene and an amino acid for immobilization on a gold surface.

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Protokół

1. Synthesis of Biotin-Fc-cysteine (1)

  1. Solid phase methods to produce resin-bound 1.
    1. Place biotin loaded resin (250 mg, 0.145 mmol) into a fritted syringe and swell the resin by drawing up dimethylformamide (5 ml) and shaking the syringe on a lab shaker for 20 min. Expel the solution and repeat dimethylformamide swelling one more time.
    2. Remove the Fmoc protecting group by adding 4-6 ml of 20% piperidine in dimethylformamide to the syringe followed by 10-15 min of shaking. Repeat the deprotection process with another 4-6 ml of piperidine. Wash the resin with a sequence of 3x dimethylformamide, 3x dimethylformamide:methanol (1:1), 3x methanol:dichloromethane (1:1), 3x dichloromethane, ~5 ml each. Do a ninhydrin test (+) on a small sampling (~10) of the beads to confirm successful deprotection by the presence of blue upon heating.
    3. Mix a solution containing 1’-Fmoc-amino-ferrocene-1-carboxylic acid (203.3 mg, 0.4350 mmol), 1-hydroxybenzotriazole hydrate (58.8 mg, 0.413 mmol), diisopropyl carbodiimide (0.0673 ml, 0.435 mmol), diisopropyl ethyl amine (0.0757 ml, 0.435 mmol), and a 4:1 mixture of dichloromethane and dimethylformamide. Draw this into the fritted syringe and gently shake on a lab shaker for 6 hr. Then expel the solution from the syringe and wash as previously described.
    4. Perform the ninhydrin test (-) as described above to confirm coupling. The ninhydrin test can still be useful in confirming coupling despite the orange color of the bead derived from the attachment of the iron containing moiety.
    5. Then remove the Fmoc group by the addition of 20% piperidine in dimethylformamide and washed as described above. The ninhydrin test (+) should be used to confirm Fmoc removal.
    6. Prepare a solution composed of Fmoc-Cys(Trt)-OH (254.8 mg, 0.4350 mmol), 1-hydroxybenzotriazole hydrate (58.8 mg, 0.4125 mmol), diisopropyl carbodiimide (0.0673 ml, 0.4350 mmol), diisopropyl ethyl amine (0.0757 ml, 0.4350 mmol), and a 4:1 mixture of dichloromethane and dimethylformamide. Add this cysteine coupling cocktail the fritted syringe and gently shake for 6 hr. Wash using the protocol described previously.
    7. Confirm coupling using the ninhydrin test (-), followed by removal of the Fmoc component with 20% piperidine and washing. Verify the free terminal amine using the ninhydrin test (+).
  2. Cleavage of 1 from the resin.
    1. Make a solution of TFA (9.45 ml), water (0.25 ml), 1,2-ethanedithiol (0.25 ml), and triisopropyl silane (0.1 ml), add it to the syringe and shake gently for 4 hr.
    2. Collect the resulting red-brown solution in an Eppendorf tube and evaporate the TFA slowly using a stream of air.
    3. Add cold diethyl ether (~15 ml) to the Eppendorf tube to precipitate 1, which will form gentle agitation. Isolate the product via centrifugation (1 g, 5 min). Then repeat cycles of diethyl ether washings (~60 ml total) and centrifuge to obtain 1 as a red/brown solid.

2. Characterization and Analysis of 1

  1. Confirm that the identity matches the connectivity and composition shown in Figure 2 using 1H (16 scans) and 13C NMR (512 scans) in deuterated methanol (300 µl) and ESI-MS analysis.
    Expect the following results:
    1H NMR spectrum (CD3OD) δ/ppm: 1.407-1.684 (m, 6H), 2.245 (t, 2H), 2.665-3.150 (m, 12H), 4.015 (t, 1H), 4.104 (d, 2H), 4.274 (q, 1H), 4.426 (d, 2H), 4.479 (q, 1H), 4.595 (t, 1H) and 13C NMR spectrum (CD3OD) δ/ppm: 24.644 (CH2), 25.472 (CH2), 28.051(CH2), 28.300 (CH2), 35.474 (CH2), 38.698 (CH2), 39.241 (CH2), 39.717 (CH2), 55.340/55.538 (Cp-ring), 60.286 (CH), 61.964 (CH), 62.521/62.821 (Cp-ring), 66.038/66.170 (Cp-ring), 69.153/69.328 (Cp-ring), 71.468/71.593 (Cp-ring), 76.466 (CH), 171.770 (C=O), 175.361 (C=O).
    ESI-MS (m/z): Found: 639.00 [1+Na]+, Theoretical: 639.1 [1+Na]+ and HR-MS (m/z): Found: 617.2049 [1+H]+ , Theoretical: 617.1622 [1+H]+.
  2. Perform HPLC, elemental analysis to confirm the composition of isolated 1.
    Carry out HPLC chromatograms using a C8 reversed phase column with 100% MeOH at a flow rate of 0.5 ml/min. Note: HPLC retention times were: 3.198-4.674 min.

3. Immobilization of 1 on a Gold Surface

  1. Cut polymer backed gold slides into squares of ~0.25 in2.
  2. Fill a 50 ml beaker with a DI water solution of 1 (~1 mM).
  3. Add the gold slide to the beaker and cover with a watch glass. Allow the glass slide to incubate O/N at RT with no agitation.
  4. Remove the gold slide from the solution and allow it dry in air.
  5. Obtain scanning electron microscopy images using a scanning electron microscope (or equivalent) to observe immobilized 1.

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Wyniki

The resin bound form of 1 is shown in Figure 2. The covalent attachment of the ferrocene component gives rise to an orange tint to the resin beads that is persistent with continuous washing and indicative of an immobilized iron containing complex as opposed to iron absorption by the PEG component of the resin bead. The resin-free form of 1 is identical in color to the resin beads. Following removal of the compound from the resin-beads, the purity and yield (68%) resultin...

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Dyskusje

The synthesis of asymmetric ferrocene derivatives is challenging in solution. For example, attempts to produce 1 in solution resulted in low yields of the desired product (less than 20%). Likewise, reactions utilizing 1’-amino-ferrocene carboxylic acid (sans Fmoc) and resin bound biotin resulted in insoluble product consistent with the polymerized product reported by Baristic et al. and minimal product.47 This is further complicated by ferrocene and its derivatives bein...

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Ujawnienia

The authors have nothing to disclose.

Podziękowania

KG was supported by the R.A. Welch Foundation grant P-1760, TCU Andrews Institute of Mathematics & Science Education (to KG), TCU Research and Creativity Activity Grant (to KG) and TCU SERC Grant (to JHS).

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Materiały

NameCompanyCatalog NumberComments
Biotin Novatag ResinNovaBiochem8550510001
TORVIQ 10 ml Luer Lock Fritted SyringeFisherNC9299151
piperidineAcrosP/3520/PB05
ninhydrin testSigma-Aldrich60017-1ea
1’-Fmoc-amino-ferrocene-1-carboxylic acidOmm ScientificSpecial Order
N,N′-DiisopropylcarbodiimideSigma-AldrichD125407-5G
Fmoc-Cys(Trt)-OHNovabiochem8520080025
trifluoroacetic acidSigma-AldrichT5408
1,2-ethanedithiolSigma-Aldrich2930
triisopropyl silaneSigma-Aldrich233781
Eppendorf tubes (20 ml)any source
methanolany sourcedry with molecular sieves prior to use & store in 100 ml media bottle for easy usage
dichloromethaneany sourcedry with molecular sieves prior to use & store in 100 ml media bottle for easy usage
dimethylformamideany sourcedry with molecular sieves prior to use & store in 100 ml media bottle for easy usage
centrifugeany source

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Keywords FerroceneBiotinBioconjugateBiosensorSolid phase SynthesisEarly Disease Detection

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