用超常光透射法定量测定人血清中的心脏标志物

The overall goal of this technique is to measure biomarkers present in a complex biological fluid background at clinically relevant concentrations in a point of care setting without the need for elaborate optical or electrical setups. This method can help in making biosensors based on extraordinary transmission a reality in point of care scenarios. The main advantage of this technique is that it doesn't need any complicated electrical or optical set-ups.

In our experiments it could reliably detect biomarkers at clinically relevant concentrations. Fabrication of the sensor chip is performed in the clean room and cannot be filmed. For demonstration purposes the procedure is represented here by an animation.

For preparation of the nickel mold coat a 220 nanometer thick layer of negative electron-beam resist onto a 600 micron thick four inch silicon wafer. Write the designed nano-hole array on this wafer, using an electron beam lithography system. To accelerate the electron beam writing write the patterns with a low dot map of 20, 000 for each 300 micron field size.

Develop the resist by immersing the four inch silicon wafer in the developer solution for 10 seconds and letting the wafer dry in air. Deposit a seed layer of a metal, such as nickel, copper, or aluminum on the silicon wafer. Then electroplate the wafer in a plating system within a nickel-sulfamate bath in two steps.

Separate the nickel mold from the silicon substrate by apply gentle, mechanical force. Next, using a nanoimprinter, imprint the nano patterns on a four inch glass wafer that has been coated with a 300 nanometer thick later of photo-curable nanoimprinting lithography resist. Transfer the mold, the photo resist, and the glass wafer to a UV light curing system and photo-cure.

If all steps have been followed correctly, the nickel mold should easily be de-molded from the photo-resist. After performing a blank etch of the photo-resist on the glass substraight, on the glass wafer, in an electronbeam deposition machine deposit a layer of chromium for metal adhesion and a layer of gold for the plasmonic sensor. Perform lift-off of the photo-resist by oxygen plasma etching for three minutes, followed by a 15 second sonofication step in acetone.

Then, dice the sample into five millimeter by five millimeter chips. The nano-hole array will occupy the central, two millimeter by two millimeter square of the chip. To acquire the data, set up the apparatus to make the optical measurements, such that a beam of white light exiting through the end of the transmitter optical fiber is columnated and is incident of the sensor surface at 90 degrees.

Light is transmitted through the whole nano-hole array. Collect the transmitted signal with the receiver optical fiber and record it with a UV-visible spectrometer operating within the range of 300 to 1, 000 nanometers. For the sensor bulk sensitivity test, deposit the standard, refractive index liquid into the liquid cell with the refractive indexes varying from 1.31 to 1.39.

Immerse the sensor chip in the standard refractive index liquid and align it with the beam of white light. Then obtain the transmission spectrum. Clean the sensor chip after each measurement with a surface active cleaning reagent and dry it with nitrogen gas.

For the sensor surface modification, clean the sensor chips by sequential immersion in isopropanol, acetone, and deionized water prior to any chemical modifications. Then, dry the sensor chips are room temperature in a steam of dry nitrogen gas. Next, incubate the sensor chips in an ethanolic solution for 12 hours at room temperature.

This will form an amine reactive, self-assembly monolayer. Following incubation, use ethanol to thoroughly rinse and dry the chips at room temperature. Next, immerse the chips into a mixture of 75 millimolar sulfo n hydroxysuccinimide and 15 millimolar EDC for 15 minutes.

This will activate the carboxsilic group of the self-assembly mono-layer. Then spot 50 microliters of a 200 microgram per milliliter anti-triponent antibody solution made in a Ph 4.5 acitate buffer onto the sensor's surface. After incubating for 30 minutes, deactivate the unreactive esters by immersing the sensor chip in one molar ethanolamine HCL solution for 15 minutes.

Finally, rinse the chip with deionized water and dry it in a stream of dry nitrogen gas at room temperature. Block any nonspecific binding by spotting 100 microliters of 1%bovine serum albumine solution onto the sensor chip's surface. Incubate for 15 minutes.

Rinse the sensor chips three times in phosphate buffered saline solution. Insert the chip into the measurement cell to record the transmission spectrum. This is the reference spectrum.

Then spot 50 microliters of the cardiac troponin one standard onto the chip's surface. Incubate the chip in a moist environment for 30 minutes. After rinsing the sensor chips three times in PBS solution, insert it into the measurement cell to record the transmission spectrum.

This is the after-binding spectrum. Next, submerge the chips in 50 millimolar glycine HCL for one minute and then rinse in PBS solution three times to regenerate the chip's surface. Measure the transmission spectrum in PBS to verify the success of the regeneration step.

The change in transmission spectrum of the biosensor upon interacting with 30 nanograms per milliliter of human cardiac troponin in serum is shown. The blue plot represents before the interaction, while the red plot represents after the interaction. The dotted circle indicates the band being tracked.

Shown here is the shift in wavelength for band two at troponin concentrations of 2.5 nanograms per milliliter, 7.5 nanograms per milliliter, 30 nanograms per milliliter, and 75 nanograms per milliliter. The error bars represent the standard deviation. This plot shows the shift in wavelengths of bands observed after regeneration for a nano-hole biosensor chip.

Shifting of the position of band two back to its original position, indicates that the regeneration step was successful. Once mastered, the measurement can be made in one hour if performed properly. While measuring bioanalytes in this method, it is important to use freshly prepared reagents.

Please make sure to eliquate out your consumables. After watching this video, you should have a good understanding of how to fabricate biosensors that operate on the principle of extraordinary optical transmission and can monitor bioanalytes against a complex background. Generally, individuals new to this method will struggle because it is difficult to generate high fidelity nano-hole arrays which make it possible to interface the chip with an optical fiber without needing any microscope and opting reproducible resins.

This technique enables you to make serial measurements which are crucial in medical settings. The fabrication of the chip goes a long way in ensuring the reproducibility of the measurements. With the basic platform remaining the same, further improvements to this technique will come from using the technique with better resolution which will improve sensitivity and from using signal processing methods to attain better signal to noise ratios.

The implication of this technique extend towards monitoring multiple bioanalytes against a complex background of serum in a point of care setting in real time.