Archives of Virology and Infectious Diseases  ( ISSN : 2994-824X )

Research Article - Volume 1, Issue 1 (2023)

A Study on Colloidal Gold-Based Immunochromatographic Assay: Monoclonal Antibodies

Moloney Pareek1, Maestro Jacob2*, Lammers Shukla3

1Nanotechnology Characterization Laboratory, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, NCI at Frederick, Frederick, MD 21702, USA

2Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, PA 15213, USA

3Health Effects Laboratory Division, National Institute of Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, WV 26505, USA

Correspondence to: Maestro Jacob, Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, PA 15213, USA. E-mail:

Received: March 02, 2023; Accepted: March 21, 2023; Published: March 28, 2023

Citation: Pareek M, Jacob M, Shukla L. Colloidal Gold Nanoparticles: The Outer boundary of Coffee-Stain Ring. Arch Virol Infect Dis. 2023;1(1):15-23.

Copyright: © 2023 Jacob M. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Droplet drying techniques showed in several studies that nanoparticles typically self-assemble in a very coffee-stain ring once complete evaporation of the solvent. Suspension comprises of assorted shapes and sizes in suspension dried on SiO2 substrate at temperature leaves behind a coffee-stain-like ring. High-resolution scanning microscope (HRSEM) pictures for at numerous locations of the coffee-stain ring show some fascinating self-assembling behavior of assorted shapes of the gold nanostructures. At outer edges of the deposited ring, side-by-side assembly of nanorods showed that such assembly begins with a skinny nanorod and ends with thick ones. just in case of thick-thin rods and spheres with comparatively dense concentration permits simply thick rods ensemble whereas alternative determined in disorder all over inside the ring. Theoretical nano- interactions conjointly affirms experimental results.


 Self-assembly; Gold nanorods; Coffee-stain ring; Shape-separation.


Self-assembly of nanoparticles is important for constructing materials which will be involve to acknowledge the collective properties of the ensuing superstructures, with uses in optics, physical science, and chemical change [1-3] these applications are heaps totally different from those of the building blocks [4-6]. Recently, several categories of nanoparticle superstructures are achieved exploitation numerous schemes, for example static forces, biomolecular recognition, and entropic effects [7-16]. Over the past few years, it's been shown that combination of assorted shapes will vastly enhance the section behavior of the resultant superstructures. Even so, there exist heaps of area to much overcome the constraints that always hinder the interpretation of these concepts into experimental realization. Moreover, understanding and manipulation of self-assembly of nano entities at the side of form and size separation still wants noticeable thought.

A sessile drop as well as nano entities in suspension once dried on a substrate typically kind a hoop like structure, referred to as “coffee-stain ring”. exploitation optical magnifier, it's been determined that upon evaporation of solvent, suspended particles in a very drop migrate towards the 3-section contact-line, consequently develop the capillary flow mechanism that ends up in the formation of ring-stain ring. Evaporation driven self-assembly is immensely necessary for the explanation that it's easy, cheap, and one can do numerous ranges (nano to micro) of assembled arrays.

At present, structural diversity of nanoparticles is increasing and therewith curiosity is additionally growing, to grasp their self-assembling still as self-separation behavior for example, experiments have discovered that nanorods and nanospheres will self-assemble into stripes like superstructures at the proximity of the border of the coffee-stain ring. Consequently, establish phase-separate result in suspension still intrinsically deposits demonstrate order to disorder transition mutually move from outer to inner fringe of the coffee-stain ring.

We show here that numerous sizes of gold nanorods in suspension preferentially self-organize principally at the border of the coffee stain ring. 2 totally different techniques area unit utilized to look at and perceive their self-assembly behavior. 1st we have a tendency to synthesize gold nanorods that comprise of assorted sizes in suspension. second 2 totally different ratio (AR3 and AR6) nanorods area unit mixed in a very outline portion. the previous showed assembly of side-by-side arrays of gold nanorods that principally begins with an agent rod and ends with a thicker rod set at the border of the coffee-stain ring. The later but shows thick nanorods assembly whereas skinny nanorods and spheres mixture didn't assemble. Such results area unit explained in terms of concentration and potential interactions at the nanoscale.



Cetyltrimethylammonium bromide (CTAB, Aldrich, 98%), element tetrachlorocuprate (HAuCl4•3H2O, 99.999%, Aldrich), caustic (AgNO3, 99%, Acros), water-soluble vitamin (AA, 99%, Merck), atomic number 11 borohydride (NaBH4, 99%, Aldrich), and acid (HCl, 37%, Merck) were all used as received while not additional purification. All water that was utilized in the synthesis was of Milli-Q quality (18.2 MΩcm), made in a very Simplicity 185 system (Millipore).


One step synthesis technique was used for creating of gold nanorods with numerous sizes. First, answer of ten milliliter (0.2M) CTAB in water was placed at 30°C for a minimum of 0.5 associate degree hour to dissolve CTAB fully in water. Then twenty-five of HAuCl4 (0.1 M) was introduce with continuous starring followed by twenty-seven (0.1 M) of AA with two hundred two hundred (0.1 M) NaOH. Finally, 50 μL (0.01M) of AgNO3 was value-added and left for nightlong at temperature.

We used the 2-step seed mediate protocol, as explained by Nikoobakht and El-Sayed to provide gold nanorods for this study. Initially, CTAB-coated seed answer was ready by adding twenty-five of HAuCl4 (0.1 M) in one0 milliliter of CTAB (0.1 M). At that point, sixty of cold NaBH4 (0.1 M) was value-added with unendingly stirring for a minimum of five min. This answer was quickly turned brownness, indicating the formation of gold seeds. The ensuing answer was unbroken at 25°C for two h to realize saturation of the seed formation.

For ratio (AR) three gold nanorods, growth answer was ready by introducing fifty of HAuCl4 (0.1 M) in one0 milliliter of CTAB (0.1 M). The ensuing answer was heated at 35°C for thirty min with slow stirring to dissolve CTAB and was left to cooldown to 25°C. At this temperature, twenty-five of AgNO3 (0.1 M) was introduced and so followed by seventy of water-soluble vitamin (0.1 M) with gentle stirring; this answer quickly turned colorless. Then, a hundred and fifty a hundred and fifty of HCl (1 M) was value-added and hydrogen ion concentration of the answer was maintained at three. Lastly, twenty-four of the seed answers was introduced into the expansion answer. This answer was left uninterrupted nightlong at two °C.

In the same approach for AR6 nanorods, growth answer was ready by adding fifty of HAuCl4 (0.1 M) in one0 milliliter of CTAB (0.1 M). This answer was unbroken at 35°C for thirty min to fully dissolve CTAB. Then keeping the answer at 25°C, twenty of AgNO3 (0.1 M) was introduced. At that point, seventy of water-soluble vitamin (0.1 M) was value-added into the expansion answer that is followed by one00 a hundred of HCl (1 M). Lastly, twenty-four of seed answer was value-added into the expansion answer and was left undisturbed nightlong at temperature.

All ready suspensions were centrifuged at 15000 rates for ten min to require out the surplus CTAB. Similarly, an equivalent growth answer (AR3) were centrifuged once more at 5600 rates for five min to separate nanospheres from the nanorods. The supernatant comprising principally nanorods was cautiously separated from the settled precipitate within the bottom of the centrifuge tube; the latter includes of principally spheres. These nanorod suspensions were hold on within the white goods.

For drop-casting depositions, SiO2 substrates were ultrasonically washed in H2O for ten min at temperature. Followed by the additional cleanup of the substrates, that were rinsed double with H2O and dried in a very element flow. Suspensions droplets (5 μL) of every answer were placed on the clean SiO2 substrate and allowed to evaporate at temperature. Nearly inside three hours the solvent is totally disappeared, going a coffee-stain ring on the surface.


Ultraviolet-visible (UV-VIS) {spectroscopy|spectrometry|spectroscopic associate degreealysis|spectrum analysis|spectrographic analysis|chemical analysis|qualitative analysis} was performed exploitation 2 totally different setups: (a) a Varian Cary three hundred Scan mass spectrometer|spectroscope|prism spectroscope} (for activity of spectra within the vary of 200-900 nm) and (a) an Ocean Optics HR2000+ spectrometer run exploitation the Spectra Suite software package together with Mikropack UV-VIS source of illumination (model DH-2000-BAL) (for the activity up to wavelengths of 1100 nm). we have a tendency to utilized customary semi micro ultraviolet radiation cuvettes with outer dimensions of twelve.5 mm × 12.5 metric linear unit × forty-five metric linear unit for the optical investigation; the optical path through the answer amounts to ten metric linear unit. Such cuvettes will delay to one.5 milliliter of answer, permitting investigation of restricted volumes of answer. UV-VIS spectrums of ready gold nanorods area unit shown in Figure 1 from the analysis of thwart wise and longitudinal peak positions within the UV–vis curves, we have a tendency to deduce that the gold nanorods concentration for black curve amounts to some 1.2 × 1010 cm-3, likewise for blue curve the densities of nanorods amounts to four.7 × 1011 cm-3. Also, for UV-VIS spectra painted by red curve contains gold nanorods that amounts to some two.1 × one010 cm-3 whereas for an equivalent red curve nanospheres variety densities adequate some two.2 × 1010 cm-3.

Figure 1: UV-VIS spectroscopic plots of synthesized gold nanorods.

High-resolution scanning microscopy (HRSEM; on a Merlin Zeiss 1550 system) was performed for imaging of our samples; typical fast voltages within the vary zero.1-30 potential unit area unit accessible. All pictures during this work were taken at two potential unit.


Suspension drop once evaporation of the solvent usually leaves behind a coffee-stain like deposits inside a fix ring. SEM pictures at 2 totally different locations within the coffee-stain ring area unit shown within the Figure 2. Red arrows indicating the direction of motion of the 3 section contact line throughout drying. At extreme right of the SEM pictures, gold nanorods assembled nicely in side-by-side fashion. shut examination of the many pictures reveal that such parallel arrangements with relevancy the drop contact line show analogous trend of aspect-by- side assembly all over inside the ring. Likewise, these initial gold nanorod deposits at the outer proximity of the contact line by and enormous self-assembled in step with their sizes. There, usually deposition originates from skinny rod and ends with a comparatively thicker rod and such deposits area unit simply includes of monolayer arrays of nanorods. moreover, mutually moves inward from the border of the coffee-stain ring, side-by-side assembled building blocks show no like orientation still as no signs of size by selection and separation. Moreover, assembly of nanorods aloof from the border seems to be no a lot of monolayer rather grows into a dense multilayered construction. SEM image at totally different location, shows fully totally different behavior aloof from the border. Likewise, tiny nanorods at the side of some sphere like particles deposit there in disordered, showing no specific trend.

Figure 2: SEM image of gold nanorod comprises of various sizes deposit in a coffee-stain ring.

Close examination of SEM pictures mostly demonstrates similar trend at the proximity of the border of coffee-stain ring as displayed in Figure 3. SEM pictures in Figures 3A and 3B show giant summary of the initial assemble arrays of gold nanorods demonstrating side-by-side assembly of 10-20 nanorods in significantly analogous manner. Such deposits area unit additional enlarged in Figures 3C and 3D, that shows self-organizations of nanorods simply at the proximity of the coffee-stain ring. Once again, demonstrating basically the similar behavior of self-assembly that begins with skinny rods and finishes with the comparatively thicker ones.

Figure 3: SEM image of gold nanorod comprises of various sizes deposit in a coffee-stain ring.

To statistically interpret such formations at the proximity of the outer finish of the coffee- stain ring, many SEM pictures area unit examined for this purpose. Histograms were premeditated for nearly two hundred rods inside the side-by-side arrays, dimension of every nanorod inside the deposited array was measured. for example, dimension of 1st nanorod that deposit on the surface was measured then second and then on up to the tenth nanorod in every assembled array. dimension distribution of 1st 5 nanorods at the border of the coffee-stain ring area unit whereas from rod variety sixth to tenth inside the similar arrays of gold nanorods area unit. The inset in the norm of the dimension of nanorod at the actual location inside the assembled array in every bar chart.

Next thick and skinny gold nanorod mixture was accustomed additional perceive such the dimensions separation impact. For this purpose, gold nanorods of AR3 that contain thick nanorods (width~12 nm) and AR6 gold nanorods that contains of skinny rods with average diameter of eight nm (width ~ eight nm). Also, AR6 suspensions contains important quantity of spheres with the gold nanorods. The ration of blending was unbroken hour of AR6 and four-hundredth of AR3 gold nanorods. SEM results for such mixture is displayed at varied locations of the coffee-stain ring. summary of 2 completely different locations at the coffee-stain rings, wherever red arrows inform towards the motion of contact line throughout drying. Each SEM pictures are displaying the similar trend, wherever nanorods self-assembled in side-by-side fashion and their long axis are minded parallel to the 3-part contact line.

In most cases inside such SEM pictures gold nanorod arrays are self-assembled within the analogous far to the length of 6-7μm as portrayed. However, inside the similar pictures far from the outer reaches, no ordered arrangements are determined. A more in-depth read of such deposits over these locations shows that these assembled arrays of gold nanorods are fashioned just by thick nanorods whereas thin-rods and spheres like nanoparticles are originated in complete disorder. moreover, thin-rod and sphere mixture found principally far from the outer-edge. However, investigation of many SEM pictures reveals that thin-rods and spheres parturition beneath the self-assembled arrays of thick nanorods. Inner deposits within the coffee-stain ring a displayed, wherever tail of side-by-side assembled thick-rods is shown within the locality of the disordered thin-rods and spheres.


The formation of self-assembly as a result of drop drying mechanism primarily establishes convictive flow towards the three-phase contact line inside the drop as a result of comparatively quicker evaporation rate at the drop ends than the middle. Consequently, such convective flow migrates nearly all suspended entities towards the three-phase contact line within the finish when complete evaporation of the binary compound part, suspension drop leaves behind a hoop like structure on the surface that just contains of nanoparticle arrays as portrayed. currently there are 2 major potentialities for the nanoparticles in suspension: (i) comparatively diluted suspension, suggests a lot of free house among particles which permit them to assemble so assemble at the shut locality of the contact line, and just in case of (ii) High density nanoparticles assembles far from the contact line and within the finish settles in varied orientations close to the sting. So, in suspension, concentration and size variations of nanoparticles appear to possess pronounced impact on self-assembly mechanism.

For instance, just in case of thick and skinny nanorods, pronounced size separation determined specially at the proximity of the outer reaches of the coffee-stain like ring. As recorded by the SEM pictures, it absolutely was determined that unremarkably side-by-side assembly of gold nanorods begins with the diluent particles and typically ends with the thicker ones. Suggesting that inside a suspension drop these nanoparticles migrate towards the three-phase contact line, wherever skinny nanorods typically within the starting of the assembled arrays showing that convective flow 1st brings diluent rods towards the sting followed by thicker particles as a result of the various rate of flow. skinny nanorods thanks to their tiny sizes flow comparatively quicker than the thick nanorods. As side-by-side array progresses towards the inner region of the coffee-stain ring, dimension of nanorods will increase virtually linearly.

Theoretically combination of engaging and repulsive interactions like depletion, Van der Waals, and electricity forces represented elsewhere between 2 nanorods are utilized on such system to grasp the formation of their self-assembled arrays. as an example, if length of a gold nanorod is thirty nm with a diameter amounts to ten nm, for these parameters the theoretical plots in that energetically favorable assembly of 2 gold nanorods is side-by-side (SS) arrangement whereas least favorable or energetically less probable position for the 2 nanorods in suspension is end-to-end (EE) arrangement. The depth of potential well {in case|just just in case} of end-to-end arrangement is almost one kT as portrayed whereas depth of such potential well deepens additional (4 kT) in case of side-by-side assembly. Therefore, such side-by-side arrangement not solely by experimentation a lot of possible however additionally agrees in theory.

Interestingly for comparatively high concentration of thick-thin gold nanorods with important quantity of spheres-like particles, solely thick rods were found assembled side-by-side parallel with the contact line whereas skinny nanorods and spheres were determined all over on the surface in chaos. It looks that skinny nanorods and spheres in suspension act like depletants that facilitate thick nanorods to assemble side-by-side. Moreover, most of those thin-rods and spheres were determined far from the outer reaches of the coffee-stain ring. Justifying our claim that these particles act like depletants and deposit far from the assemble arrays of thick nanorods.

Theoretically, the interaction energy is a lot of engaging for thick nanorods (diameter = sixteen nm) whereas just in case of skinny nanorods such energy is considerably low. Skinny nanorods the depth of potential well is around 4kT whereas just in case of thick nanorods depth of potential well will increase and having worth around six.5kT. therefore 2 thick nanorods in suspension attract each other a lot of powerfully than the skinny nanorods. As a result, chance of thick nanorods to assemble in suspension will increase as witnessed in SEM pictures displayed on top of.

It is tough for nanoparticles to induce assemble in an exceedingly dense setting that contains of 3 completely different geometries like skinny rods, thick rods, and spheres with collective concentration in suspension amounts to eight.1 × 1031 cm-3. On the premise of SEM pictures, it's evident that in an exceedingly dense setting strongest interaction survives and as a result assembles with its own kind. However, thin-rods and spheres in an exceedingly chaotic setting couldn't presumably notice free house to ensemble.


 Self-assembly of 2 completely different systems are investigated. As ready polydisperse gold nanorods assembled side-by-side close to the outer reaches of the contact line with especially size distribution. Such assembly typically begins from skinny rod and ends with comparatively thick rods. Analysis of many SEM pictures showed that because the assembled array progresses towards the inner region of the coffee- stain ring, the diameter of gold nanorods will increase. On the opposite hand, mixture of assorted shapes and sizes as a result of their dense concentration in suspension just facilitate thick nanorods whereas thin-rods and spheres were determined in disorder principally found far from the outer reaches of the coffee-stain ring. In theory, energetically most possible assembly for the 2 nanorods in suspension is side-by-side arrangement and for the thick nanorods such interactions are more practical than skinny nanorods in suspension.


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