Gold Nanopar­ti­cles

Intro­duc­tion of Gold Nanoparticles

Gold nanopar­ti­cles have been used as effec­tive car­ri­ers in lat­er­al flow assays to detect the tar­get ana­lytes in clin­i­cal sam­ples. Due to their unique phys­i­cal and chem­i­cal prop­er­ties, col­loidal gold can be eas­i­ly con­ju­gat­ed with anti­bod­ies or anti­gens with­out alter­ing their activ­i­ty or speci­fici­ty. High-qual­i­­ty gold mate­ri­als are essen­tial to deliv­er supe­ri­or con­sis­ten­cy and per­for­mance in rapid diag­nos­tic tests.

The inter­ac­tion of gold nanopar­ti­cles with proteins

Adsorp­tion of pro­teins on the sur­face of gold nanopar­ti­cles is dri­ven by three major forces : elec­tro­sta­t­ic inter­ac­tion, hydropho­bic bind­ing, and dative bond­ing. Ini­tial­ly, the neg­a­tive­ly charged gold par­ti­cles will attract pos­i­tive­ly charged func­tion­al groups in pro­teins. As the pro­teins approach, hydropho­bic patch­es with­in them bind to the hydropho­bic areas of the gold par­ti­cles through hydropho­bic inter­ac­tions. Addi­tion­al­ly, pro­teins with sul­­fur-con­­tain­ing amino acid residues may form Au-S bonds with gold atoms. There­fore, the for­ma­tion of gold nanopar­ti­­cle-pro­tein com­plex­es is a com­plex process that depends on the char­ac­ter­is­tics of both the pro­teins and the gold nanopar­ti­cles. The shape, size, and sur­face chem­istry of gold play impor­tant roles in this process.

Char­ac­ter­is­tics of gold nanoparticles

The phys­i­cal prop­er­ties of gold nanopar­ti­cles affect the effi­cien­cy and repro­ducibil­i­ty of their con­ju­ga­tion with pro­teins, which, in turn, affect assay sen­si­tiv­i­ty, speci­fici­ty and man­u­fac­tur­ing con­sis­ten­cy. For lat­er­al flow appli­ca­tions, the ide­al gold nanopar­ti­cles should have the fol­low­ing attributes :

Monodis­per­si­ty

Monodis­per­si­ty is a key per­for­mance para­me­ter for col­loidal gold. Monodis­perse gold nanopar­ti­cles have a uni­form size and spher­i­cal shape. When a lat­er­al flow test is run, gold con­ju­gates, along with the clin­i­cal sam­ple, will flow even­ly from the con­ju­ga­tion pad onto the mem­brane. How­ev­er, if the gold par­ti­cles are poly­dis­perse, with dif­fer­ent sizes and irreg­u­lar shapes, the larg­er gold con­ju­gates will move slow­er than the small­er ones on the mem­brane. This dif­fer­ence in flow rate can cause vari­abil­i­ty in the test results.

Size

The size of gold nanopar­ti­cles direct­ly affects the sen­si­tiv­i­ty of lat­er­al flow assays. In sand­wich immunoas­says, larg­er gold nanopar­ti­cles (60nm & 70nm) can achieve high­er assay sen­si­tiv­i­ty than small­er ones (30nm & 40nm).

Left is the sen­si­tiv­i­ty com­par­i­son of five dif­fer­ent sizes of gold nanopar­ti­cles (WI-30, WI-40, WI-50, WI-60, WI-70) in pro­cal­ci­tonin (PCT) assay by test­ing human serum samples.

(A)30nm (WI-30); (B)40nm (WI-40); (C)50nm (WI-50); (D)60nm (WI-60); (E)70nm (WI-70)

Col­loidal stability

Gold nanopar­ti­cles are com­mon­ly syn­the­sized by cit­rate reduc­tion method. Due to the lim­i­ta­tions of this method, the dis­per­sion sta­bil­i­ty decreas­es when the size of col­loidal gold reach­es 50nm or larg­er. Poor­ly pre­pared gold nanopar­ti­cles tend to be poly­dis­perse and may grad­u­al­ly aggre­gate over time.

Typ­i­cal­ly, high-qual­i­­ty gold nanopar­ti­cles have a shelf life of at least 12 months under appro­pri­ate con­di­tions. It is rec­om­mend­ed that man­u­fac­tur­ers car­ry out sta­bil­i­ty stud­ies to assess the changes in gold mate­ri­als over extend­ed peri­ods of time before pro­ceed­ing to the

test strip pro­duc­tion stage.

High-con­­cen­­tra­­tion gold par­ti­cles (> 50 OD) offer sev­er­al advan­tages over con­ven­tion­al ones (1-5 OD) for lat­er­al flow appli­ca­tions. Con­ju­gat­ing anti­bod­ies to gold par­ti­cles at a

Con­cen­tra­tion

high con­cen­tra­tion could improve cou­pling effi­cien­cy, as it increas­es the chances of anti­bod­ies attach­ing to the sur­face of gold. Addi­tion­al­ly, this approach could min­i­mize the reac­tion vol­ume in the gold con­ju­ga­tion pro­ce­dure and save time and costs asso­ci­at­ed with cen­trifu­ga­tion steps afterward.

Our gold prod­ucts are sup­plied at a con­cen­tra­tion of 100 OD, allow­ing kit man­u­fac­tur­ers to per­form anti­body con­ju­ga­tion at 40 OD. This sig­nif­i­cant­ly reduces the cost of reagents (anti­body or anti­gen) by up to two-thirds with­out sac­ri­fic­ing sen­si­tiv­i­ty, mak­ing it a cost-effec­­tive solu­tion for lat­er­al flow applications

Gold Con­ju­ga­tion Protocol

The pro­to­col pro­vides gen­er­al guide­lines for con­ju­gat­ing anti­bod­ies or pro­teins to gold nanopar­ti­cles. As pro­teins vary in their net charge and charge dis­tri­b­u­tion, it is nec­es­sary to deter­mine the opti­mal con­ju­ga­tion con­di­tions, includ­ing buffer type, pH, pro­tein con­cen­tra­tion and incu­ba­tion time, through a pre­lim­i­nary exper­i­ment before pro­ceed­ing with the con­ju­ga­tion procedure.

Reagents :

• Old nanopar­ti­cles at a con­cen­tra­tion of 100 OD
• Anti­bod­ies or pro­teins sup­plied at a con­cen­tra­tion
  of >1mg/​mL
• Gold con­ju­gates stor­age buffer con­tain­ing buffer salts,
  sur­fac­tants, sug­ars (sucrose or tre­halose), and block­ing
  pro­teins (BSA or Casein)
• 10% (w/​v) NaCl solution
• Buffer at the required pH

Pro­to­col

Step 1 : Screen­ing buffer type and pH for conjugation

The most com­mon­ly used buffers for con­ju­ga­tion are list­ed below. In our stan­dard pro­ce­dure, the cou­pling of pro­teins to gold nanopar­ti­cles is car­ried out in a set of unique buffers with dif­fer­ent pH points. The opti­mal pH and buffer type are then deter­mined by salt-induced aggre­ga­tion test in step 2. The fol­low­ing is an exam­ple of cou­pling mouse mon­o­clon­al anti­bod­ies to gold nanopar­ti­cles under var­i­ous buffer conditions.

Pro­ce­dure :

1. Add 60 μL of the fol­low­ing four buffers with the required pH to each of the four test tubes.
A. 0.01M Phos­phate buffer, pH 6.8
B. 0.01M Phos­phate buffer, pH 7.4
C. 0.01M Borate buffer, pH 7.4
D. 0.01M Borate buffer, pH 8.0

Note : As phys­i­cal and chem­i­cal prop­er­ties can vary sig­nif­i­cant­ly between pro­teins, we sug­gest exper­i­ment­ing with addi­tion­al buffer types and pH for your proteins.

A list of can­di­date buffer solu­tions is pro­vid­ed below. Typ­i­cal­ly, the pH for con­ju­ga­tion should be slight­ly above the iso­elec­tric point of the bind­ing pro­teins. This is con­sid­ered to be a start­ing point for deter­min­ing of the appro­pri­ate pH. The opti­mal bind­ing pH may require fur­ther fine-tun­ing using the select­ed buffer type.

2. Label each tube with all per­ti­nent infor­ma­tion.
3. Vor­tex gold nanopar­ti­cles (100 OD) to ensure that the par­ti­cles are com­plete­ly sus­pend­ed.
4. Pipette 40 uL of gold nanopar­ti­cles (100 OD) into each tube that con­tains the four select­ed buffer solu­tions with the required pH.
5. Mix and thor­ough­ly vor­tex the dilut­ed gold nanopar­ti­cles (40 OD).
6. Add the appro­pri­ate amount of anti­body to each labeled test tube. The typ­i­cal con­cen­tra­tion for con­ju­ga­tion in step 1 is 60 ug/​mL.
7. React for 2 hours at room tem­per­a­ture with con­tin­u­ous mix­ing.
8. After con­ju­ga­tion, take a small aliquot (25 uL) of the gold con­ju­gates (40 OD) for fur­ther saltin­duced aggre­ga­tion test in step 2.

Step 2 : Salt-induced aggre­ga­tion test*

The salt-induced aggre­ga­tion test is a quick and sim­ple method to mon­i­tor the col­loidal sta­bil­i­ty of gold con­ju­gates. Briefly, after con­ju­ga­tion, mix the gold con­ju­gates with a NaCl solu­tion and leave them at room tem­per­a­ture for 5 min­utes. A col­or change from red to pur­ple or blue indi­cates the aggre­ga­tion of the gold con­ju­gates below, sug­gest­ing that the buffer type and pH may not be suit­able for your anti­bod­ies or proteins.

Salt-induce aggre­ga­tion test

Pro­ce­dure :

1. Add 975 uL of deion­ized water into each of the four clean test tubes.
2. Pipette 25 uL of gold con­ju­gates (40 OD) into each test tube con­tain­ing deion­ized water, to make a final vol­ume of 1 mL (Note : The con­cen­tra­tion of gold con­ju­gates is now 1 OD). Label the tubes with all per­ti­nent infor­ma­tion.
3. Add 100 uL of 10% (w/​v) NaCl solu­tion to each test tube con­tain­ing the dilut­ed gold con­ju­gates (1 OD).
4. Mix and leave at room tem­per­a­ture for 5 min­utes. Observe the col­or change of the gold con­ju­gates.**
5. Read the results. Select the buffer type and pH that result in the best col­loidal sta­bil­i­ty of gold con­ju­gates.
* To deter­mine the opti­mal buffer type and pH, the most accu­rate method is to val­i­date the gold con­ju­gates direct­ly in func­tion­al test strips. Select the buffer type and pH that result in opti­mal sen­si­tiv­i­ty, speci­fici­ty, and sta­bil­i­ty for your assays.
** Use 1 OD of gold nanopar­ti­cles as a col­or con­trol. This can be done by dilut­ing 100 OD of gold nanopar­ti­cles with deion­ized water.

Step 3 : Deter­min­ing the opti­mal con­cen­tra­tion of bind­ing pro­teins to gold nanoparticles

1. Add 60 uL of the select­ed buffer with the required pH to each of the four test tubes.
2. Pipette 40 uL of gold nanopar­ti­cles (100 OD) into each tube and mix thor­ough­ly.
3. Add the appro­pri­ate amount of anti­body to each tube to achieve final con­cen­tra­tions of 40 ug/​mL, 50 ug/​mL, 60 ug/​mL and 80 ug/​mL, respec­tive­ly.*
4. React for 2 hours at room tem­per­a­ture with con­tin­u­ous mix­ing.
5. Add 5 uL of 10% (w/​v) BSA solu­tion to the gold con­ju­gates (40 OD). Mix thor­ough­ly and leave at room tem­per­a­ture for 1h.**

6. After block­ing, cen­trifuge at 2310 g for 10 min. The time and speed of the cen­trifu­ga­tion depend on the size of gold nanopar­ti­cles and should be adjust­ed
accord­ing­ly for opti­mal per­for­mance.
7. Remove the super­natant and resus­pend gold con­ju­gates to a final con­cen­tra­tion of 40 OD in gold con­ju­gates stor­age buffer.
8. Take an aliquot of gold con­ju­gates (40 OD) for fur­ther func­tion­al test­ing. Select the anti­body con­cen­tra­tion that result in opti­mal sen­si­tiv­i­ty, speci­fici­ty, and sta­bil­i­ty for your assays.

Step 4 : Deter­min­ing the opti­mal incu­ba­tion time

1. Add 60 uL of the select­ed buffer with the required pH to each of the four test tubes.
2. Pipette 40 uL of gold nanopar­ti­cles (100 OD) into each tube and mix thor­ough­ly.
3. Add the appro­pri­ate amount of anti­body deter­mined in step 3 to each of the four test tubes.
4. React at room tem­per­a­ture for 30min, 60min, 90min and 120min, respectively.

5. After con­ju­ga­tion, take a small aliquot (25 uL) of gold con­ju­gates (40 OD) for fur­ther salt-induced aggre­ga­tion test in step 2.
6. Repeat step 2 to deter­mine the opti­mal incu­ba­tion time.*
* To deter­mine the opti­mal incu­ba­tion time, the most accu­rate method is to val­i­date the gold con­ju­gates direct­ly in func­tion­al test strips. Select the incu­ba­tion time that result in opti­mal sen­si­tiv­i­ty, speci­fici­ty, and sta­bil­i­ty for your assays.

Step 5 : Gold con­ju­ga­tion with pro­teins under appro­pri­ate conditions

1. Add 60 uL of the select­ed buffer with the required pH to the test tube.
2. Pipette 40 uL of gold nanopar­ti­cles (100 OD) into the tube and mix thor­ough­ly.
3. Add the appro­pri­ate amount of the anti­body and react at room tem­per­a­ture for the opti­mal time deter­mined in step 4.
4. Add 5 uL of 10% (w/​v) BSA solu­tion to the gold con­ju­gates (40 OD). Mix thor­ough­ly and leave at room tem­per­a­ture for 1h.*

5. After block­ing, cen­trifuge at 2310 g for 10 min. The time and speed of the cen­trifu­ga­tion depend on the size of gold nanopar­ti­cles and should be adjust­ed accord­ing­ly for opti­mal per­for­mance.
6. Remove the super­natant and resus­pend gold con­ju­gates to a final con­cen­tra­tion of 40 OD in gold con­ju­gates stor­age buffer.
7. Mix thor­ough­ly and store at 4℃ for fur­ther use.

* The opti­mal block­ing agents need to be deter­mined exper­i­men­tal­ly for each assay. BSA, Casein, or oth­er com­mer­cial block­ers are com­mon­ly used in lat­er­al flow applications