Appendix must me explained to the patient before

Appendix A

Preparation of gold-nano-particles

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The materials needed for the synthesis of gold-nano-particles are as follows: (i) sodium citrate (Mallinckrodt, Analytical Reagent), (ii) polyviny-1-alcohol) denoted PVA (Polysciences, hydrolysis 99.0-99.8 mol%), (iii) NaBH, (Alfa Inorganic), (iv) HAuCl (Strem Chemicals), and the (iv) dyes (Eastman Kodak Co., laser grade). MiliQ water is needed for dissolving all contents.


Preparation of solutions

The following two different Au sol preparations could be used


Procedure (a)


Initially a 100 ml solution prepared solution using 5×10-3 M HAuCl and was added to 300 ml of ice-cold 2×10-3, M NaBH4, solution and vigorous stirred. 50 ml solution of 1% PVA was added during the reduction reaction. After completion of the reaction, the mixed solution was heated for 1 hour to decomposition of excess NaBH4. The final volume was made up to 500 ml.


Procedure (b)


240 mg of HAuC1 was dissolved in 500 ml of water and the solution was allowed to boil. Then 50 ml of 1% sodium citrate was added and it was boiled for 1 hour.


The gold nano particle containing solution prepared by procedure (a) were purple in color and those prepared by procedure (b) were wine red in color, but they both had absorption maxima at approximately 530 nm. The concentrations of gold (Au) is given in gram-atoms per liter.









Appendix B

Collection of tear samples on a Shirmer strip

Hands were washed properly before performing the test. The patient was positioned comfortably with head supported. All kind of distractions must be avoided during handling the patient. A good lighting facility must be available. The procedure must me explained to the patient before performing the test.


Reasons for Schimer’s test


To collet protein content present in tear sample of the diseased individual.


Materials needed


(i) Schirmer’s test strips

(ii) Watch or clock

(ii) Clear adhesive tape

(iv) Pen

(v)  PBS solution (1x)

Application of any anesthetic drop or other eye medication must be avoided before performing the test. This may give a false result.




Initially two strips were taken out from the sterile packet and labeled as ‘R’ (right) and ‘L'(Left). Then each strip was bended at the notch to a 90-degree angle. The patient was asked to look with an index finger and the lower eyelid was gently pulled down. The bent end of the strip was hooked over the centre of the lower eyelid and allowed to be seated inside. The procedure was repeated for the other eye. Then the patient was asked not to squeeze, but just to keep the eyes gently closed. After five minutes, the patient was asked to open both eyes and look upwards. Both strips were carefully removed. Using the package scale, the length of the moistened area on the strip was measured from the notch and it was indicated with a pen mark. The 1ml of 1X PBS solution was taken and strips were dipped into it in different eppendorf tubes and kept at -80ºc before performing any experiment.