gold nanocolloids have attracted strong interest from scientists for over a century and are now being heavily investigated for their potential use in a wide variety of medical and biological applications . for example , potential uses include surface - enhanced spectroscopy , biological labeling and detection , gene - regulation , and diagnostic or therapeutic agents for treatment of cancer in humans . their versatility in a broad range of applications stems from their unique physical , chemical , and surface properties , such as : ( i ) size - and shape - dependent strong optical extinction and scattering at visible and near infrared ( nir ) wavelengths due to a localized surface plasmon resonance of their free electrons upon excitation by an electromagnetic field ; ( ii ) large surface areas for conjugation to functional ligands ; and ( iii ) little or no long - term toxicity or other adverse effects in vivo allowing their high acceptance level in living systems . these new physical , chemical , and surface properties , which are not available from either atomic or bulk counterparts , explain why gold nanocolloids have not been simply chosen as alternatives to molecule - based systems but as novel structures which provide substantive advantages in biological and medical applications . the prerequisite for most of intended biological and medical applications of gold nanoparticles is the further surface modification of the as - synthesized gold nanoparticles via conjugation of functional ligand molecules to the surface of the gold nanoparticles . the surface functionalization of gold nanoparticles for any biological or medical applications is crucial for at least two reasons . first is control over the interaction of the nanoparticles with their environment , which is naturally taking place at the nanoparticle surface . appropriate surface functionalization is a key step to providing stability , solubility , and retention of physical and chemical properties of the nanoparticles in the physiological conditions . second , the ligand molecules provide additional and new properties or functionality to those found inherently in the core gold nanoparticle . these conjugated gold nanoparticles bring together the unique properties and functionality of both the core material and the ligand shell for achieving the goals of highly specific targeting of gold nanoparticles to the sites of interest , ultra - sensitive sensing , and effective therapy . nowadays , the major strategy for surface modification of gold nanoparticles include coating gold nanoparticles with polymer , for example peg containing reactive functional groups , such as — cooh and — nh 2 , which are ready for the conjugation of targeting ligands . however , current strategy of coating gold nanoparticles with functional groups , such as — cooh and — nh 2 , often results in highly charged surfaces , which promote their binding to biomolecules in the biological systems through ionic interactions , causing nanoparticles to aggregate in biological environments and thus exhibit strong non - specific binding to various cells and tissues that is undesirable in many in vitro and in vivo applications . in the present invention , provided are methods which permits versatile and controllable surface modification of gold nanoparticles with both positive charged peg and negative charged peg ( e . g ., for addressing the issues and challenges described above ) and provide methods to fabricate gold nanoparticles having minimum cellular uptake by macrophage cells via forming zwitterionic surface containing both positive and negative charges on the same gold nanoparticle . it is believed that , prior to the present invention , there was no way to conjugate a defined number of positive charged ligands and negative charged ligands onto the surface of gold nanoparticles to control the surface charge ( or zeta potential ) for optimizing their biomedical performance . as discussed above , the overwhelming majority of gold nanoparticles are prepared by the standard sodium citrate reduction reaction . this method allows for the synthesis of spherical gold nanoparticles with diameters ranging from 5 to 200 nanometers ( nm ) which are capped with negatively charged citrate ions . the capping controls the growth of the nanoparticles in terms of rate , final size , geometric shape and stabilizes the nanoparticles against aggregation by electrostatic repulsion . in contrast to the prior process of bottom - up fabrication using wet chemical processes , in certain embodiments , the gold nanoparticles used in the present invention are produced by a top - down nanofabrication approach . the top - down fabrication methods of the present invention start with a bulk material in a liquid and then break the bulk material into nanoparticles in the liquid by applying physical energy to the material . the physical energy can be mechanical energy , heat energy , electric field arc discharge energy , magnetic field energy , ion beam energy , electron beam energy , or laser beam energy including laser ablation of the bulk material . the present process produces a pure , bare colloidal gold nanoparticle that is stable in the ablation liquid . the ablation liquids comprise a plurality of solvents selected from but not limited to deionized water , methanol , ethanol , acetone , and dimethylformamide . the present invention is noted limited by the top - down nanofabrication approach used in the present invention . these methods involve the generation of the nanoparticles from the bulk material in the presence of the suspension medium . in one embodiment the process comprises a one step process wherein the application of the physical energy source , such as mechanical energy , heat energy , electric field arc discharge energy , magnetic field energy , ion beam energy , electron beam energy , or laser energy to the bulk gold occur in the suspension medium . the bulk source is placed in the suspension medium and the physical energy is applied thus generating nanoparticles that are immediately suspended in the suspension medium as they are formed . in another embodiment the present invention employs a two - step process including the steps of : 1 ) fabricating gold nanoparticle arrays on a substrate by using photo , electron beam , focused ion beam , nanoimprint , or nanosphere lithography as known in the art ; and 2 ) removing the gold nanoparticle arrays from the substrate into the suspension liquid using one of the physical energy methods . tabor , c ., qian , w ., and el - sayed , m . a ., journal of physical chemistry c , vol 111 ( 2007 ), 8934 - 8941 ; haes , a . j . ; zhao , j . ; zou , s . l . ; own , c . s . ; marks , l . d . ; schatz , g . c . ; van duyne , r . p . journal of physical chemistry b , vol 109 ( 2005 ), 11158 . in both methods the colloidal gold is formed in situ by generating the nanoparticles in the suspension medium using one of the physical energy methods . in certain embodiments of the present invention , colloidal suspensions of gold nanoparticles are produced by pulsed laser ablation of a bulk gold target in deionized water as the suspension medium . fig1 schematically illustrates a laser - based system for producing colloidal suspensions of nanoparticles of complex compounds such as gold in a organic liquid using pulsed laser ablation . in certain embodiments , a laser beam 1 is generated from an ultrafast pulsed laser source , not shown , and focused by a lens 2 . the source of the laser beam 1 can be a pulsed laser or any other laser source providing suitable pulse duration , repetition rate , and / or power level as discussed below . the focused laser beam 1 then passes from the lens 2 to a guide mechanism 3 for directing the laser beam 1 . alternatively , the lens 2 can be placed between the guide mechanism 3 and a target 4 of the bulk material . the guide mechanism 3 can be any of those known in the art including piezo - mirrors , acousto - optic deflectors , rotating polygons , a vibration mirror , or prisms . preferably the guide mechanism 3 is a vibration mirror 3 to enable controlled and rapid movement of the laser beam 1 . the guide mechanism 3 directs the laser beam 1 to a target 4 . in one embodiment , the target 4 is a bulk gold target . the target 4 is submerged a distance , from several millimeters to preferably less than 1 centimeter , below the surface of a suspension organic liquid 5 . the target 4 is positioned in a container 7 additionally but not necessarily having a removable glass window 6 on its top . optionally , an o - ring type seal 8 is placed between the glass window 6 and the top of the container 7 to prevent the liquid 5 from leaking out of the container 7 . additionally but not necessarily , the container 7 includes an inlet 12 and an outlet 14 so the liquid 5 can be passed over the target 4 and thus be re - circulated . the container 7 is optionally placed on a motion stage 9 that can produce translational motion of the container 7 with the target 4 and the liquid 5 . flow of the liquid 5 is used to carry the nanoparticles 10 generated from the target 4 out of the container 7 to be collected as a colloidal suspension . the flow of liquid 5 over the target 4 also cools the laser focal volume . the liquid 5 can be any liquid that is largely transparent to the wavelength of the laser beam 1 , and that serves as a colloidal suspension medium for the target material 4 . in one embodiment , the liquid 5 is deionized water . the system thus allows for generation of colloidal gold nanoparticles in situ in a suspension liquid so that a colloidal gold suspension is formed . the formed gold nanoparticles are immediately stably suspended in the liquid and thus no dispersants , stabilizer agents , surfactants or other materials are required to maintain the colloidal suspension in a stable state . in work conducted during the development of embodiments of the present invention , the following laser parameters were used to fabricate gold nanocolloids by pulsed laser ablation of a bulk gold target in acetone : pulse energy of 10 uj ( micro joules ), pulse repetition rate of 100 khz , pulse duration of 700 femtoseconds , and a laser spot size on the ablation target of about 50 um ( microns ). for the preparation of au nanocolloids a 16 mm ( millimeter ) long , 8 mm wide , and 0 . 5 mm thick rectangular target of au from alfa aesar was used . for convenience , the au target materials can be attached to a bigger piece of a bulk material such as a glass slide , another metal substrate , or a si substrate . more generally , for the present invention the laser ablation parameters are as follows : a pulse duration in a range from about 10 femtoseconds to about 500 picoseconds , preferably from about 100 femtoseconds to about 30 picoseconds ; the pulse energy in the range from about 1 μj to about 100 μj ; the pulse repetition rate in the range from about 10 khz to about 10 mhz ; and the laser spot size may be less than about 100 μm . the target material has a size in at least one dimension that is greater than a spot size of a laser spot at a surface of the target material . work conducted during the development of embodiments of the present invention found that stable colloidal solution of gold nanoparticles with only partial surface modification could be fabricated . also , the surface coverage amount of functional peg molecules on the surface of the gold nanoparticles can be tuned to be any percent value between 0 and 100 %. fig2 shows the physical characteristics of aunps (˜ 20 nm in diameter based on tem imaging ) after mixing with hs - peg - cooh ( thiolated negative charged peg molecule terminated with carboxyl group at its distal end ) with different hs - peg - cooh / aunp molar ratios from zero to 2000 . this molar ratio range was chosen based on the surface area of 20 - nm aunps and the footprint of peg molecules . for pegylation we selected thiolated peg 5000 with molecular weight of 5000 daltons , which is most widely used to modify gold nanoparticles . the thiol concentration (& gt ; 95 %) was confirmed by an ellman &# 39 ; s test . during pegylation , the au nanoparticle concentration was fixed at 1 . 0 nm , determined by correlating our measured extinction spectra to the experimentally determined extinction cross - section data ( 8 . 8 × 10 8 m − 1 cm − 1 for aunps with a diameter of 20 nm ). fig2 a and 2 b show the optical spectra of aunps at all hs - peg - cooh / aunp ratios and the corresponding optical density ( od ) at the absorption peak , respectively . the overlapping curves and the constant od reveal that there is no loss of aunps after pegylation for each of the hs - peg - cooh / aunp molar ratio . the high colloidal stability of laser - made aunps after pegylation with different molar ratios is probably attributed to the highly negatively charged surface as displayed in fig2 c , which shows that the zeta potential is independent of the hs - peg - cooh / aunp molar ratio . although there are not any capping ligands on the aunp surfaces before pegylation , the aunps made by femtosecond pulse laser ablation have a natural negative surface with zeta potential of about − 33 . 3 mv . the negatively charged surface is caused by partial oxidation from the oxygen present in solution , followed by proton transfer to adjacent hydroxide ions . the pegylation process was further demonstrated by monitoring the change in particle size after pegylation with increasing hs - peg - cooh / aunp molar ratios , as shown in fig2 d . the data show that the hydrodynamic layer thickness consistently increased with increasing molar ratios of hs - peg - cooh / aunp , and reached a constant at 26 nm after the aunp surface was saturated at molar ratio of 500 . this reveals an increase in peg chain density on aunp surfaces upon increasing the hs - peg - cooh / aunp molar ratio . for free peg molecules in a good solvent , the radius of gyration r g can be empirically calculated by : r g = 0 . 181 × n 0 . 58 where n is the number of ethylene glycol ( eg ) monomer units per peg chain . for the peg 5000 used here , r g is 2 . 8 nm and the size increase should be around 11 . 2 nm ( 2 . 8 × 4 ) if the peg chains form isolated hemispheres ( mushroom ) on the surface with a critical peg density σ *( 1 / πr g 2 ) around 0 . 04 peg / nm 2 . at molar ratio of 500 the peg density σ is around 0 . 4 peg / nm 2 , which is 10 times higher than the critical grafting density . the grafting peg chains are most likely reconfigured , stretching out in a brush conformation as shown in the insert of fig1 d . this explains why the hydrodynamic layer thickness ( δd ) is more than four times the radius of gyration . the differential peg - cooh chain density on aunps after pegylation at varying molar ratios of hs - peg - cooh / aunp was detected by centrifuging the solutions and re - suspending the pellet of aunps . for molar ratios less than 100 the pellet could not be re - dispersed after centrifugation , as shown in fig3 a , revealing the peg - cooh chain density is too low to maintain a stable formulation . as the molar ratio increases from 100 to 200 the absorption spectra show that more aunps are recovered after centrifugation , determined by the increase of the absorption peak at 524 nm . however , detection of a second , smaller peak ( or ‘ bump ’) at ˜ 650 nm suggests these partially pegylated aunps tend to form aggregates after centrifugation , indicating a higher peg - cooh chain density is required to maintain stability . fig3 b shows the od at the absorption peak of aunps pegylated with hs - peg - cooh at different molar ratios . the data clearly show which pegylated aunp formulations are stable after centrifugation , based on the peg density on the aunp surfaces . as the molar ratio increases from 100 to 300 the od number increases by 25 times , with a relative change from 4 % to 100 % when compared to the od before centrifugation , respectively . the data also indicate that even though the aunp surfaces are not fully covered with peg at a molar ratio of 300 , the aunps are stable against centrifugation . furthermore , surface modification was carried out on gold nanoparticles with hs - peg - nh 2 ( thiolated positive charged peg molecule terminated with amine group at its distal end ). fig4 shows the physical characteristics of aunps (˜ 20 nm in diameter based on tem imaging ) after mixing with hs - peg - nh 2 with different hs - peg - cooh / aunp molar ratios from zero to 2000 . the aunp concentration decreases after incubation at a range of molar ratios from 200 to 500 . for the molar ratio of 300 , a dip was observed in the od for the peak wavelength of the absorption spectra after incubation with aunps as shown in fig4 a and 4 b . it is worth noting that the color of the solutions at these ratios does not change to purple and there are no bumps on any of the absorption spectra including that for a molar ratio of 300 , revealing that the loss of the aunps was not because of aggregation of aunps . it was found that , at these molar ratio ranges , aunps become stuck on the eppendorf tube ( observing that the inside of the tube is red ). as the molar ratio of hs - peg - nh 2 / aunp increases from 50 to 300 , the zeta potential of pegylated aunps transforms from highly negative (− 44 . 9 mv ) to slightly positive (+ 14 . 4 mv ) as shown in fig4 c . as previously mentioned , at a molar ratio of 300 the aunps are only 60 % fully covered . this data suggests that two factors attribute to the dip formation observed in fig4 b : a zeta potential close to zero and partial pegylation . as discussed below , this unstable range can be avoided by addition of peg molecules with opposite charges ( e . g ., hs - peg - cooh ) prior to adding , for example , hs - peg - nh 2 . as the molar ratio of hs - peg - nh 2 / aunp further increases to 500 the zeta potential reaches + 52 . 2 mv and becomes constant when the molar ratio is higher than 500 , indicating successful pegylation with increasing peg - nh 2 chain density on the aunps until the surfaces become saturated . successful pegylation was also demonstrated by monitoring the size increase after pegylation with increasing hs - peg - nh 2 / aunp molar ratios , as shown in fig4 d . the data show that the hydrodynamic layer thickness increases by 26 nm at the saturated hs - peg - nh 2 / aunp molar ratio of 500 , which is consistent with the pegylation using hs - peg - cooh . the data combined clearly reveal a controlled increase in the peg - nh 2 chain density as one increases the hs - peg - nh 2 / aunp molar ratio . using conjugation of gold nanoparticles with hs - peg - nh 2 molecules as an example , worked conducted during the development of embodiments of the present invention further analyzed the efficiency of pegylation using a fluorescamine - based assay to quantify the concentration of free hs - peg - nh 2 in the supernatant . in this assay a non - fluorescent fluorescamine reacts with a primary amine to generate a compound that emits fluorescence at 480 nm when excited at 390 nm . originally developed for quantification of primary amines in biomolecules , this assay has sensitivity on the pm scale . in doing this experiment , the hs - peg - nh 2 concentration was varied during incubation from 50 to 2000 nm while keeping the aunp concentration constant at 1 . 0 nm . therefore , the initial input molar ratio between hs - peg - nh 2 and gold nanoparticle ranges from 50 to 2000 . fig5 a and 5 b show the fluorescence spectra and the corresponding calibration curve that correlates the fluorescence intensity at 480 nm to the concentration of free hs - peg - nh 2 without mixing with aunps . a linear relationship was found in the concentration range of 50 to 2000 nm of hs - peg - nh 2 . this assay was then applied to measure the free hs - peg - nh 2 in the supernatants after incubation with aunp at different molar ratios followed by centrifugation , shown in fig5 c and 5 d . although the hs - peg - nh 2 concentration increases 10 times up to 500 nm , there is no obvious fluorescence increase after measuring the supernatants until the pegylation becomes saturated ( at molar ratio of 500 ). this reveals that most of the added hs - peg - nh 2 bind to aunps without any detectable free hs - peg - nh 2 in the supernatants . measuring the peg concentration in supernatants to estimate the peg chain density on aunps is a widely used method . when the ratio is higher than 500 one begins to see the fluorescence increase in the supernatant , indicating that the aunps gradually become saturated . this result is consistent with the zeta potential measurements . these combined experiments demonstrate a highly efficient pegylation process that allows control of the peg density on aunps by manipulating the molar ratio between hs - peg - cooh or hs - peg - nh 2 and aunp , as long as the ratio is less than the saturated one . based on controllable stable conjugation of hs - peg - cooh molecules and hs - peg - nh 2 molecules on surface of gold nanoparticles described above with the surface coverage amount of hs - peg - cooh molecules and hs - peg - nh 2 molecules on the surface of the gold nanoparticles can be tuned to be any percent value between 0 and 100 %. as such , the present invention allows methods of fabricating gold nanoparticles with zwitterionic surface , which permits precisely control of number of both negative charged ligands and positive charged ligands bound onto surface of gold nanoparticles . in certain embodiments , this method comprises : performing surface modification of gold nanoparticles for forming zwitterionic surface by conjugation of both negative charged ligands and positive charged ligands onto surface of colloidal gold nanoparticles in a sequential manner . exemplary steps include the follows ; step 1 : the negative charged ligands are first mixed with said colloidal suspension of gold nanoparticles at room temperature for at least 30 minutes . step 2 : addition of positive charged ligands to colloidal suspension of gold nanoparticles also at room temperature . the total amount of negative charged ligand added to the said colloidal gold nanoparticles is no more than the minimum amount required to provide a monolayer of bound negative charged ligand to the total of colloidal gold nanoparticles based on a footprint of negative charged ligand bound on gold nanoparticles and total amount of positive charged ligand added to the colloidal gold nanoparticles is no more than the minimum amount required to bind to all free binding sites left on surface of colloidal gold nanoparticles after conjugation of negative charged ligands onto surface of colloidal gold nanoparticles . step 3 : after mixing , the mixture is kept undisturbed for 24 hours at room temperature to provide a sufficient amount of time for both positive charged ligands and negative charged ligands to be conjugated onto the surfaces of colloidal au nanoparticles ; and optionally , after step 2 and before step 3 , adding to colloidal gold nanoparticles more said negative charged ligands to ensure saturation of said binding sites on surface of said colloidal gold nanoparticles for maximizing colloidal stability of said colloidal gold nanoparticles . as an example , work conducted during the development of embodiments of the present invention used the above method to fabricate gold nanoparticles with zwitterionic surface containing both hs - peg - cooh molecules and hs - peg - nh 2 molecules . the schematic illustration of fabrication of gold nanoparticles with zwitterionic surface bearing both peg - cooh molecules and peg - nh 2 molecules on their surface in a sequential manner is shown in fig6 . first partially pegylating aunps with hs - peg - cooh at room temperature for at least 30 minutes , then pegylating with hs - peg - nh 2 . as long as the aunps are not saturated ( e . g ., the molar ratio between combined number of hs - peg - cooh and hs - peg - nh 2 and number of au nanoparticles is lower than about 500 ), one can control the density of both hs - peg - cooh and hs - peg - nh 2 covalently attached to aunps . at least 30 minutes after addition of hs - peg - nh 2 , than adding more hs - peg - cooh to saturate the surface for maximum aunp stability . finally , collection of fabricated gold nanoparticles with zwitterionic surface by centrifuge at 15000 g for 30 minute , removing the supernatant , and then redispersing into a phosphate buffered saline ( pbs ) with a ph of 7 . 4 prior to their use in cell experiments . in addition , power of fabricated gold nanoparticles with zwitterionic surface could be also formed after collection of them from solution . in work conducted during the development of embodiments of the present invention , a series of colloidal solutions of gold nanoparticles with zwitterionic surface have been made using method as shown in the fig6 . the molar ratio between the number of hs - peg - cooh and the number of gold nanoparticles is fixed at 150 and the molar ratio between the number of hs - peg - nh 2 and the number of gold nanoparticles ranges from 0 to 300 . the stability of these obtained gold nanoparticles after redispersed into phosphate buffered saline ( pbs ) with a ph of 7 . 4 is examined and the results are shown in the fig7 . for all obtained gold nanoparticles , they are very stable . two hours after being redispersed into pbs buffer , the decrease of surface plasmon resonance of gold nanoparticles is less than 40 %. thus , the method could fabricate gold nanoparticles having zwitterionic surface containing both hs - peg - cooh and hs - peg - nh 2 with their surface coverage could be controlled to be any amount between 0 and 100 %, which are stable in phosphate buffered saline . the term “ stable ” means that the decrease of absorbance intensity caused by localized surface plasmon resonance of gold nanoparticles at 518 to 530 nm , more specifically at 520 nm two hours after redispersing gold nanoparticles into pbs buffer is less than 40 % compared to the absorbance measured at time zero ( immediately after redispersing ). next , non - specific uptake of obtained gold nanoparticles was tested with zwitterionic surface by macrophage cells . as shown in fig8 , the data show that as the amount of peg - nh 2 on aunps increases from 50 to 300 , the non - specific uptake of au / cell ( pg ) decreases by half from 0 . 63 ± 0 . 01 to 0 . 33 ± 0 . 01 . the reduced cell uptake is likely due to the overall surface charge becoming more neutral with addition of more peg - nh 2 . as shown in the table 1 , with fixed amount of peg - cooh , the increase of amount of peg - nh 2 do increase the zeta potential of gold nanoparticle from − 48 mv , − 35 mv , − 17 mv , and to − 2 mv as increasing amount of peg - nh 2 per gold nanoparticle from 0 to 500 . table 1 shows the zeta potential of gold nanoparticles with zwitterionic surface as function of number of peg - cooh molecules and peg - nh 2 molecules bound onto surface of gold nanoparticles . the number of peg - cooh per gold nanoparticle is fixed at 150 and the number of peg - nh 2 ranges from 0 to 900 . furthermore , these aunps with zwitterionic surface have significant less non - specific uptake than the control aunps decorated with a single type of peg - r ( i . e . — och 3 , — cooh , — nh 2 ). for instance , the double - charged aunps show a relative uptake ranging from 40 % to 76 % when compared to aunp - peg - och 3 , the control with the least uptake . various chemical functional groups , such as thiol , amine , disulfide , and phosphine , possess a high affinity for the surface of gold nanoparticles . thiol groups are considered to show the highest affinity for gold surfaces , approximately 200 kj / mol , and therefore a majority of gold nanoparticle surface functionalization occurs through using ligand molecules having thiol groups which bind to surfaces of gold nanoparticles via a thiol - au bond . in addition to peg or poly ( ethylene oxide ) ( peo ) polymer , other polymers having molecular weight in the range of from 200 daltons to 100 , 000 , 000 daltons selected from but not limited to poly ( 2 -( methacryloyloxy ) ethyl phosphorylcholine ), poly ( 2 -( dimethylamino ) ethyl methacrylate ), poly ( acrylic acid ), and poly ( ethylene glycol ) containing at least one functional group having an affinity for surface of said gold nanoparticles could also be used as functional ligand . in certain embodiments , the au nanoparticles have the shape of a sphere , rod , prism , disk , cube , or core - shell structures , cages , and frames , wherein they have at least one dimension in the range of from 1 to 200 nm ( e . g ., 1 . . . 50 . . . 100 . . . 150 . . . or 200 nm ). in addition , the method of surface modification described in this invention can be used for structures ( e . g ., nanostructures ) which have outer surfaces that are only partially covered with gold . thus , while only certain embodiments have been specifically described herein , it will be apparent that numerous modifications may be made thereto without departing from the spirit and scope of the invention . further , acronyms are used merely to enhance the readability of the specification and claims . it should be noted that these acronyms are not intended to lessen the generality of the terms used and they should not be construed to restrict the scope of the claims to the embodiments described therein . it is intended that the invention not be limited by the specific embodiments and their variations and combinations as described herein - above .
the present invention relates to methods , compositions , and kits for generating conjugated gold nanoparticles . in certain embodiments , the present invention provides methods of generating unsaturated conjugated gold nanoparticles by mixing naked gold nanoparticles with a first type of attachment molecules at a molar ratio such that the attachment molecules attach to the naked gold particles at a density level below the saturation level of the naked gold particles . in some embodiments , a second type of attachment molecules are mixed with the unsaturated conjugated gold nanoparticles to generate double - conjugated gold nanoparticles .