Hypothermal opto-thermophoretic tweezers

Optical tweezers have profound importance across fields ranging from manufacturing to biotechnology. However, the requirement of refractive index contrast and high laser power results in potential photon and thermal damage to the trapped objects, such as nanoparticles and biological cells. Optothermal tweezers have been developed to trap particles and biological cells via opto-thermophoresis with much lower laser powers. However, the intense laser heating and stringent requirement of the solution environment prevent their use for general biological applications. Here, we propose hypothermal opto-thermophoretic tweezers (HOTTs) to achieve low-power trapping of diverse colloids and biological cells in their native fluids. HOTTs exploit an environmental cooling strategy to simultaneously enhance the thermophoretic trapping force at sub-ambient temperatures and suppress the thermal damage to target objects. We further apply HOTTs to demonstrate the three-dimensional manipulation of functional plasmonic vesicles for controlled cargo delivery. With their noninvasiveness and versatile capabilities, HOTTs present a promising tool for fundamental studies and practical applications in materials science and biotechnology.


Introduction
The development of optical tweezers has led to tremendous advances in many fields such as optical nanomanufacturing, 1 microrobotics, 2 cell mechanics, 3 and nanomedicine. 4 Optical tweezers trap target objects by the gradient force, which depends on the refractive index, particle size, and laser wavelength. 5 High laser power is usually required to trap nanomaterials and biological objects that have a low refractive index contrast with their surroundings, which can induce damage to the materials and reduce the cell viability. 6 To overcome these challenges, different variations of optical tweezers, such as plasmonic tweezers, 7-10 opto-electronic tweezers, 11,12 and opto-acoustic tweezers, 13 have been developed. However, they are usually limited by specific substrates, complex setups, or confined working ranges. Recently, optothermal tweezers have been developed to achieve versatile manipulation of colloidal particles under a light-controlled temperature gradient [14][15][16][17][18] . While optothermal tweezers enable enhanced trapping capability with a laser power that is 2-3 orders of magnitude lower than optical tweezers, 19 optical heating can cause thermal stress and degradation to the particles and biological cells. In addition, since many colloids and cells show a thermophobic behavior and move away from the laser heating spot, optothermal tweezers require additional surfactants or salts to tune their thermophoretic response. [20][21][22][23] However, trapping cells and biological objects in required fluidic environments is often essential for biological applications to reduce the effect of additives and elucidate the bio-physiochemical interactions of the cells. [24][25][26] In this work, we propose hypothermal opto-thermophoretic tweezers (HOTTs) to overcome these limitations. Specifically, we couple environmental cooling and localized laser heating to achieve low power thermophoretic trapping of target objects and simultaneously avoid optical and thermal damage.
More importantly, this cooling strategy also plays a vital role in facilitating the thermophilic behavior to enable the trapping of diverse colloids at different conditions. We also demonstrate the successful trapping and manipulation of fragile erythrocytes in different tonicities to resemble different biophysio-chemical functionalities. We further show the capability of HOTTs for three-dimensional manipulation (3D) of plasmonic vesicles for light-controlled drug delivery.

Working Principle of HOTTs
Figs. 1a and 1b depict the general schemes of opto-thermophoretic trapping at ambient temperature and under environmental cooling, respectively. A thermoplasmonic substrate is used to generate a temperate gradient (∇ ) under local laser heating (see Methods). The particle under the temperature gradient is subject to a thermophoretic force ( th ), 20,27 which can be expressed as where is the Soret coefficient of the particle, is the Boltzmann constant, and is the average temperature around the particle (see Supplementary Note 1). The direction of thermophoretic force is dependent on the sign of and a positive (or negative) leads to a repulsive (or attractive) force.
is a function of many parameters, including colloid composition, ionic concentrations, surface effects, particle size, and temperature. 28,29 decreases with the decreasing temperature in the majority cases, which can be described by an empirical equation 30 where ,∞ is the high-temperature limit, * is the transition temperature where changes the sign, and 0 represents the strength of the temperature effect. At the ambient temperature, is positive for most objects and the thermophoretic force repels them away from the laser (Figure 1a). In HOTTs, we adopt an environment-cooling strategy to enable a negative and a thermophoretic attractive force to trap the particle at the hotspot (Figure 1b). A custom temperature controller based on Peltier cooling is designed to enable fast cooling of the sample ( Figure S1 and Supplementary Note 2). As a proof-of-concept demonstration, we compared the trapping behaviors of 1 m polystyrene (PS) microparticle in deionized (DI) water at two different conditions. At the ambient temperature of 27 0 C, the PS microparticle was repelled away from the laser beam due to a net repulsive thermophoretic force ( Figure 1c). In contrast, when the environmental temperature was cooled down to 4 0 C, the particle was successfully trapped at the laser beam center by the thermophoretic attraction force (Figure 1d and Supplementary Video 1).
Figure 1: Working principle of HOTTs: a) At ambient temperature, thermophoretic force ( ℎ ) repels the particle away from the laser in most conditions. White arrows indicate ℎ decomposed along and perpendicular to the substrate b) In HOTTs, ℎ becomes attractive to trap particles at a sub-ambient temperature. c) Schematic and timelapse optical images showing the repelling of a 1 m PS particle in DI water by the laser beam at an ambient temperature of 27 0 C d) The same particle was trapped at the laser beam at a sub-ambient temperature of 4 0 C. The green crosshair indicates the laser beam center. Laser wavelength: 532 nm, laser power: 40 W, beam radius: 850 nm, scale bars: 2 m.

Versatility of HOTTs
Next, we demonstrate the use of HOTTs to trap diverse microparticles in different conditions to demonstrate its wide applicability ( Figure 2, Figure S2, and Supplementary Video 2-4). In all cases, thermophoretic trapping of colloids (e.g., PS and silica microparticles) is enabled or significantly enhanced in HOTTs at a reduced ambient temperature. We measured the trapping stiffness to examine the trap strength at different temperatures (Supplementary Note 3). Figure 2a shows the trajectories of a 1 m PS particle trapped at varying temperatures. As the temperature reduces, the particle becomes more confined with respect to the laser beam center. Figure  ). It is noted that the Soret coefficient of the particle is tuned from 0.1 K -1 to -2 K -1 after reducing the temperature from 27 0 C to 4 0 C. The direction of thermophoretic force inverts and causes attraction, and the magnitude of the force increases more than 10-fold, which contributed to the increased trapping stiffness.
The thermophoretic response of the particles also depends on the colloidal concentration, 31  Trapping stiffness dependence on sample temperature at single-particle concentration indicates the enhancement of trapping efficiency at lower temperatures for varying laser powers (i-iii), sizes (ii, iv), materials (ii, v), and solutions (i-iii, iv-v). c,d) Optical images of repulsion at 27 0 C (yellow panels) and trapping at 4 0 C (blue panels) of 1 m PS particle in DI water at a high concentration of 28.6 mg/mL (c) and low concentration (d) of 0.29 mg/mL. Laser power is 50 W. e) Trapping probability of 1 m PS particles as a function of sample temperature and colloidal concentration. The highest concentration of 1 m PS particles at 100% relative concentration is 28.6 mg/mL. Laser power is 50 W. Scale bars: (a) 1 m (c,d) 5 m.

Trapping of erythrocytes in distinct tonicities using HOTTs
Erythrocytes (also known as erythrocyte cells or red blood cells) are important biological entities that are currently used in drug delivery 33,34 and disease diagnostics. 35,36 Optical trapping of erythrocytes has promoted the understanding of cell mechanics and cell-cell interactions. 37 However, these studies are based on the local photo-deformation of the cell membrane and are limited to mature erythrocytes in isotonic solution only. Erythrocytes in different tonicities (hypertonic and hypotonic) serve as potential markers for pathophysiological disease diagnostics like sickle cell anemia 38 and malaria. 39 The tonicity of extracellular fluid alters the shape and size of erythrocytes, which has been recently used to determine the severity of illness caused in SARS-COV-2 patients. 40

3D manipulation of plasmonic vesicles and controlled cargo release
Extracellular and synthetic vesicles have shown great importance in bioimaging, drug delivery, biological transport processes, and therapeutics. [44][45][46] Plasmonic vesicles are gold-coated vesicles with controlled optical and spectroscopic properties for diverse biomedical applications. [47][48][49][50] Although optical and optothermal trapping of naked vesicles or gold nanoparticles has been achieved, 51,52 optical trapping of plasmonic vesicles is challenging due to the large plasmon-enhanced scattering force because of the gold layer. Also, the heat produced during laser illumination causes an uncontrollable thermophoretic force which usually directs the vesicle towards the cold (away from the laser beam) at ambient temperature. 53 In addition, the heat generated also induces drug release from plasmonic vesicles. The capability of trapping while maintaining the integrity of plasmonic vesicles will enable precise positioning followed by optically triggered drug release, and it holds great promises in several applications.
Here, we demonstrate the trapping and 3D manipulation of plasmonic vesicles by HOTTs, followed by controlled cargo release using a dual laser beam setup. A 660 nm laser beam is utilized to manipulate the vesicle, while a 532 nm laser beam is utilized to rupture the vesicle. Under 660 nm laser irradiation, the gold coating on the plasmonic vesicles absorbs light to generate a highly localized temperature gradient across the vesicle (Figure 4a), which creates a self-induced thermophoretic force on the vesicle. 54 Meanwhile, the high optical scattering force causes the vesicle to repel away from the focus of the laser beam at the ambient temperature irrespective of the thermophoretic force direction ( Figure   4b and Supplementary Video 8). Meanwhile, the trapping of plasmonic vesicles can be achieved by HOTTs at a sub-ambient temperature of 4 0 C. In this case, the self-induced thermophoretic force becomes attractive and is greatly enhanced to overcome the optical repulsion force and enable the 3D trapping of the vesicle near the focal plane (Figure 4b). After positioning the vesicle, the subsequent illumination of a 532 nm laser beam can generate intense heat to rupture the plasmonic vesicle and release the cargo (Figure 4c).
We first showed the manipulation of a plasmonic vesicle in the vertical direction by simply tuning the focus position of the laser beam (Figure 4d). The vesicle is steadily trapped and elevated for over 55 m. Next, the vesicle is transported in the lateral plane to demonstrate the in-plane manipulation ( Figure 4e). We further demonstrated versatile 3D manipulation of plasmonic vesicles by HOTTs in highly complex and challenging environments ( Figure S4 and Supplementary Video 9). After the vesicle is transported to the target position, a 532 nm laser beam is excited to rupture the membrane to release the cargo (Calcein dye) as shown in Figure 4f (also see Figure S5 and Supplementary Video 10). We observed an increase in fluorescent intensity since the calcein dye is self-quenched in the plasmonic vesicle and emits a stronger fluorescence upon release and dilution.

Fabrication of thermoplasmonic substrate:
Glass coverslips were triple-rinsed with iso-propyl alcohol and water and cleaned under a nitrogen gun.
The coverslips were then loaded into a thermal evaporator (Kurt J Lesker Nano36), and 4.5 nm gold films are thermally deposited at a pressure of 1 x 10 -7 torr at a rate of 0.1 nm/s. Later, gold-deposited coverslips are thermally annealed at 550 0 C for two hours (ramp for 2 hours, constant temperature of 550 0 C for 2 hours, and ramp for 2 hours). For microparticle experiments, thermally annealed substrates are used as prepared after cleaning using DI water and nitrogen gun. For erythrocyte experiments, the substrates are immersed in a 1mM 11-mercaptohexanoic acid in ethanol to prevent the adhesion of red blood cells onto the substrate. The modified substrates are cleaned using water droplets (gentle cleansing) to remove excess solution for uniform functional layer formation.

Plasmonic vesicles preparation:
Plasmonic vesicles were prepared via a two-step method following the previously reported method with minor modifications. 55 First, Dipalmitoylphosphatidylcholine (DPPC) and cholesterol in a 4:1 molar ratio were dispersed in chloroform and dried with N2, followed by overnight evaporation under a vacuum. The dry lipid film was then dispersed in 10 mM PBS containing calcein 75 mM for 1 hour and subsequently extruded through 400 nm polycarbonate membranes for 11 passes using Avanti Mini Extruder (Avanti Polar Lipids). Free calcein was removed by centrifugation at 5000 g for 10 mins and then washed with PBS three times. Second, gold nanoparticles were decorated onto DPPC liposome using the in-situ gold reduction method. Aqueous solutions of gold chloride (10 mM) and ascorbic acid (40 mM) were prepared. Gold chloride solution was added and gently mixed with liposome suspension (1.5 mM lipid concentration) in a molar ratio of 1:4 until uniformly distributed, followed by the addition of the same volume of ascorbic acid solution. Following reduction, plasmonic vesicles were separated from unreacted ascorbic acid and gold chloride by centrifugation (5000g, 10 mins) and then stored at 4°C until use. For trapping experiments, plasmonic vesicles that are in the sub-micrometer regime (800-1500 nm) are used due to their easy visualization under the microscope.

Optical Setup:
For substrate-based trapping, a 532 nm laser beam (Laser Quantum Ventus 532) is passed through a 5X beam expander and directed into the objective (Nikon Plan Fluor 40x, NA 0.75) of an optical microscope (Nikon Ti-E) through a series of reflective mirrors. The liquid sample containing the target objects are loaded into a 120 m thick spacer that acts as a microchamber. A charged coupled device (CCD -Nikon DS-Fi3) is used to visualize and record particle-trapping videos. Phase camera (Phasics, SID4 Bio) is used to evaluate the temperature increase because of laser heating of the substrate. The sample is placed on an aluminum sample holder that provides edge-support on all sides of the coverslip.
The Peltier thermoelectric cooler (Laird Thermal Systems SH10-23-06-L1-W4.5) along with the aluminum heat sink is then rested on top of the glass coverslips. The weight of the heat sink ensures perfect contact of the thermoelectric cooler with the sample and the temperature resistance across the interface is assumed to be negligible. An annular cooler is selected to enable the light path through the device. A corresponding through-hole is drilled into the heat sink, for white light to travel through the sample and reach the camera.