Thermoswitchable electrokinetic ion-enrichment/elution based on a poly(N-isopropylacrylamide) hydrogel plug in a microchannel

This paper reports a novel protocol consisting of the thermomodulated electrokinetic enrichment, elution, and separation of charged species based upon a thermoswitchable swelling-shrinking property of a poly(N-isopropylacrylamide), PNIPAAm, hydrogel. A 0.2-1 mm long PNIPAAm hydrogel plug was photopolymerized inside a glass microfluidic channel to produce a composite device consisting of the PNIPAAm hydrogel plug and the glass microchannel (abbreviated as plug-in-channel). After voltage was applied to the composite device, anions, such as FITC, could be enriched at the cathodic end of the PNIPAAm plug when the temperature of the plug was kept below its lower critical solution temperature (LCST, ～32 °C). The concentrated analytes could then be eluted by electroosmotic flow when the temperature of the plug was heated above the LCST. The mechanism of the thermoswitchable ion enrichment/elution process was studied with the results presented. The analytical potential of the composite device was demonstrated for the temperature-modulated preconcentration, elution, and separation of FITC-labeled amino acids.     

Regulation of protein binding toward a ligand on chromatographic matrixes by masking and forced-releasing effects using thermoresponsive polymer

A novel concept of affinity regulation based on masking and forced-releasing effects using a thermoresponsive polymer was elucidated. Affinity chromatographic matrixes were prepared using either poly(glycidyl methacrylate-co-ethyleneglycol dimethacrylate) or poly(glycidyl methacrylate-co-triethyleneglycol dimethacrylate) beads immobilized with ligand molecule, Cibacron Blue F3G-A (CB), together with poly(N-isopropylacrylamide) (PIPAAm), a polymer with a cloud point of 32 degrees C. Two different lengths of spacer molecules were used for the immobilization of CB while maintaining the PIPAAm size constant. Chromatographic analyses using bovine serum albumin as a model protein showed a clear correlation between spacer length and binding capacity at temperatures lower than the lower critical solution temperature (LCST) of PIPAAm. The binding capacity under the LCST was significantly reduced only when the calculated spacer length was shorter than the mean size of the extended PIPAAm. Furthermore, the adsorbed protein could be desorbed (released) from the matrix surface by lowering the temperature to below the LCST while maintaining other factors such as pH and ion strength. Selective recovery of human albumin from human sera was demonstrated using this newly developed thermoresponsive affinity column.     

N-异丙基丙烯酰胺共聚物的合成及温敏性

温度敏感性高分子聚(N-异丙基丙烯酰胺)(PNIPAAm)在水溶液中有较低临界溶解温度(LCST)。文中采用两种新型温度敏感性单体甲基四氢呋喃丙烯酰胺(THFAA)和甲基四氢呋喃甲基丙烯烯酰胺(THFMA),均聚合成了亲水性较好且其LCST高于PNIPAAm的两种聚合物。用THFAA,THFMA与PNIPAAm进行自由基共聚合,研究了共聚物的组成对共聚物较低临界溶解温度的影响。结果表明,两种温度敏感性单体共聚能有效地改变共聚物的LCST,通过改变聚合时两种单体的摩尔配比制备共聚物P(NIPAAm-co-THFAA)及P(NIPAAm-co-THFMA),可以实现LCST的可控调节。     

Formulation and characterization of chitosan-based hydrogel films having both temperature and pH sensitivity

Chitosan-based hydrogel films having both temperature and pH sensitivity were prepared by blending chitosan with temperature sensitive poly (N-isopropylacrylamide) (PNIPAAm) and polyethylene glycol (PEG, Mw 2000). PEG was added to enhance film properties, such as thermal, mechanical and swelling properties. Differential scanning calorimetry (DSC) study indicated that the physically blended films exhibited a lower critical solution temperature (LCST) identical to that of pure PNIPAAm (around 32 °C). FT-IR data indicated that the temperature sensitivity is due to the PNIPAAm component in the film. The thermal analysis showed that chitosan and PNIPAAm were compatible and the blended films are apt to crystallize. The X-ray diffraction study further showed that the blended films had a higher crystallinity level than chitosan or PNIPAAm alone. The newly formed crystalline domains acted as physical crosslinkers and greatly increased the crosslinking level of the blended films, which, in turn, affected the swelling behavior and mechanical property of the blended films. Scanning electron microscopy (SEM) revealed that the blended swollen films exhibited a more porous structure at 37 °C (>LCST) than at room temperature (<LCST), though their swelling ratios were reduced as temperature increased from room temperature to 37 °C because of the dehydration nature of PNIPAAm at temperatures above its LCST. The results demonstrated that physically blended temperature sensitive films could be formulated, which are capable of producing more pores upon heating. The blended films were also found to be pH sensitive due to the fact that chitosan, one of the film components, has many pendant amino groups.     

Thermosensitive Y-shaped micelles of poly(oleic acid-Y-N-isopropylacrylamide) for drug delivery

A novel thermosensitive amphiphilic copolymer comprised of two hydrophobic poly(oleic acid) (POA) segments and one hydrophilic poly(N-isopropylacrylamide) (PNIPAAm) segment was designed and synthesized. The structure of the copolymer was confirmed as Y-shaped by FTIR, 1H NMR, and SEC-MALLS analysis. A cytotoxicity study shows that the P(OA-Y-NIPAAm) copolymer exhibits good biocompatibility. The copolymer may self-assemble into micelles in water, with the hydrophobic POA segments at the cores of micelles and the hydrophilic PNIPAAm segments as the outer shells. The resulting micelles demonstrate temperature sensitivity with a lower critical solution temperature (LCST) of 31.5 degrees C and a critical micelle concentration (CMC) of 12.6 mg L(-1). Transmission electron microscopy (TEM) shows that the micelles exhibit a nanospheric morphology within a narrow size range of approximately 10-30 nm. A study of controlled release reveals that the self-assembled micelles have great potential as drug carriers.     

Thermal-sensitive PBMA-b-PNIPAAm amphiphilic block copolymers brushes and their assembling micelles

Brush-like block copolymers with poly(t-butyl methacrylate) (PBMA) and poly(N-ispropylacrylamide) (PNIPAAm) as side arms, PBMA-b-PNIPAAm, were designed and synthesized via a simple free radical polymerization route. The chemical structure of these polymer brushes was characterized and determined by nuclear magnetic resonance (1H NMR), and Fourier transform infrared spectrometry (FT-IR). The micellar formation by these polymer brushes in aqueous solutions were detected by a surface tension technique, and the critical micelle concentration (CMC) ranged from 1.53 to 8.06 mg L(-1). The morphology and geometry of polymer micelles were investigated by transmission electron microscope (TEM) and dynamic light scattering (DLS). The polymer micelles assume the regularly-spherical core-shell structure with well-dispersed individual nanoparticles, and the particle size was in the range from 36 to 93 nm. The PNIPAAm segments exhibited a thermoreversible phase transition, so the resulting block polymer brushes were temperature-sensitive and the low critical solution temperature (LCST) was determined by UV-vis spectrometer at about 28.82-29.40 degrees C. The characteristic parameters of the polymer micelles such as CMC, micellar size and LCST values were affected by their compositional ratios and the length of hydrophilic or hydrophobic chains. The self-assembled micelles are expected to be used in specific biomedical fields as a candidate of drug controlled release carrier.     

Thermo-sensitive poly(N-isopropylacrylamide)/ mesoporous silica nanocomposites as controlled delivery carriers: loading and release behaviors for drug ibuprofen

Thermo-sensitive nanocomposites based on mesoporous silica SBA-15 and poly(N-isopropylacrylamide) (PNIPAAm) have been synthesized via in situ radical polymerization in mesopores. The resultant materials were used as carriers to construct temperature-responsive controlled drug delivery systems. Loading of model drug ibuprofen (IBU) was ascertained by IR and UV-vis/DRS spectroscopy, and the mesostructure and pore properties of the delivery system were characterized by small-angle XRD and N2 adsorption-desorption experiment. Study on drug uptake indicated that higher polymer content in the composite, higher IBU concentration in loading solution and lower loading temperature below the lower critical solution temperature (LCST) could increase the loading amount of IBU by means of interaction between IBU and polymer and trap effect of the polymer chains in pores. Different from the uptake of IBU, however, the release of drug followed a positive temperature-responsive manner, that is, the release was accelerated upon heating above the LCST, while decelerated and lasted for a longer period of time below the LCST. This feature allows the material to function as a reversible fast/slow transition switch or rate regulator responsive to environmental temperature and to be potentially interesting in controlled delivery and other smart application fields.     

Studies on micellization behavior of thermosensitive PNIPAAm-b-PLA amphiphilic block copolymers

Thermo-sensitive amphiphilic block copolymers, poly(N-isopropylacrylamide)-block- poly(D,L-lactide) (PNIPAAm-b-PLA), were synthesized through a simple free radical copolymerization route based on a bifunctional initiator, 2,2'-azobis(2-methylpropion amidine) dihydrochloride (AMAD), followed by the ring-opening polymerization of D,L-lactide in the presence of Sn(Oct)2 catalyst. The Chemical structure characterization were conducted by means of 1H NMR, FT-IR, GPC. The amphiphilic PNIPAAm-b-PLA block copolymers could self-assemble into micelles with regularly spherical shape in an aqueous solution, with a TEM diameter range of 46-56 nm, DLS hydrodynamic diameter of 158-199 nm. This behavior depends on the environmental temperature, the hydrophobic interactions among PNIPAAm molecular chains, intermolecular hydrogen bonding between the PNIPAAm chains and water molecules as well as intramolecular hydrogen bonding between -CONH2 groups. The copolymers held a CMC from 5.50 to 6.17 mg L(-1) and LCST from 31.41-32.03 degrees C, being more or less affected by their compositions, PLA or PNIPAAm block length. The as-prepared PNIPAAm-b-PLA block polymers are anticipated to be applied as a potential candidate of drug release carriers.     

温度/pH敏感性荧光胶束的制备及性能研究

通过溴乙酰溴与9-氨基吖啶(9-AA)的酰胺化反应,合成了带有2个活性溴原子的新型荧光性引发剂9-AA-Br。核磁共振氢谱(1 H-NMR)测定表明其结构明确。以氯化亚铜(CuCl)/四氮杂十四员大环冠醚(Me6[14]aneN4)为催化体系,由9-AA-Br引发N-异丙基丙烯酰胺(NIPAAm)进行原子转移自由基聚合(ATRP),成功合成了结构明确、分子量可控的双臂型PNIPAAm大分子荧光探针,由紫外分光光度计测得其最低临界溶解温度(LCST)在32℃左右,且随着溶液浓度及聚合物分子量的增加而降低。温度低于LCST时,PNIPAAm大分子荧光探针在溶液中能进行自组装形成胶束,由透射电镜(TEM)观察表明,胶束的大小在500nm左右,该聚合物胶束还具有pH敏感性,在碱性条件下随着pH的增大,荧光发射峰变强。     

Patterning enzymes inside microfluidic channels via photoattachment chemistry

We have developed a general method for photopatterning well-defined patches of enzymes inside a microfluidic device at any location. First, a passivating protein layer was adsorbed to the walls and floor of a poly(dimethylsiloxane)/glass microchannel. The channel was then filled with an aqueous biotin-linked dye solution. Using an Ar+/Kr+ laser, the fluorophore moieties were bleached to create highly reactive species. These activated molecules subsequently attached themselves to the adsorbed proteins on the microchannel walls and floor via a singlet oxygen-dependent mechanism. Enzymes linked to streptavidin or avidin could then be immobilized via (strept)avidin/biotin binding. Using this process, we were able to pattern multiple patches of streptavidin-linked alkaline phosphatase inside a straight microfluidic channel without the use of valves under exclusively aqueous conditions. The density of alkaline phosphatase in the patches was calculated to be approximately 5% of the maximum possible density by comparison with known standards. Turnover was observed via fluorogenic substrate conversion and fluorescence microscopy. A more complex two-step enzyme reaction was also designed. In this case, avidin-linked glucose oxidase and streptavidin-linked horseradish peroxidase were sequentially patterned in separate patches inside straight microfluidic channels. Product formed at the glucose oxidase patch became the substrate for horseradish peroxidase, patterned downstream, where fluorogenic substrate turnover was recorded.     

Preparation of stable dispersions of chemically reduced graphene oxide through noncovalent interactions with poly(N-isopropyl acrylamide)-grafted pluronic copolymer

Stable dispersions of chemically reduced graphene oxide (RGO) were prepared by simple noncovalent interaction with poly(N-isopropyl acrylamide) (PNIPAAm)-grafted Pluronic (NGP). Prepared NGP/RGO assembly solutions showed temperature-dependent optical absorbance change below or above their lower critical solution temperature (LCST). Interestingly, LCSTs of NGP/RGO assemblies were unaffected compared with LCSTs of pristine NGPs regardless of the degree of PNIPAAm grafting in NGPs probably because noncovalent anchoring between PNIPAAm chains and RGO plates in NGP/RGO assembly is weak. This supports that PNIPAAm grafting on Pluronic occurred preferentially in hydrophilic PEO segments because hydrophobic PPO segments of NGP must be strongly anchoring on hydrophobic surface of RGO plates while PEO segments will be extended from RGO surface to aqueous medium.     

壳聚糖/聚(N-异丙基丙烯酰胺)全互穿网络水凝胶的制备及性能

以壳聚糖(CS)和N-异丙基丙烯酰胺(NIPAAm)为原料,N,N'-亚甲基双丙烯酰胺(BIS)为交联剂,制备具有温度和pH值双敏感性的全互穿网络水凝胶(Full-IPN);利用红外光谱(FT-IR)对其分子结构进行表征,扫描电镜(SEM)观察其内部形貌,并通过DSC对其低临界溶解温度(LCST)进行表征,最后研究了不...     

Improving aptamer selection efficiency through volume dilution, magnetic concentration, and continuous washing in microfluidic channels

The generation of nucleic acid aptamers with high affinity typically entails a time-consuming, iterative process of binding, separation, and amplification. It would therefore be beneficial to develop an efficient selection strategy that can generate these high-quality aptamers rapidly, economically, and reproducibly. Toward this goal, we have developed a method that efficiently generates DNA aptamers with slow off-rates. This methodology, called VDC-MSELEX, pairs the volume dilution challenge process with microfluidic separation for magnetic bead-assisted aptamer selection. This method offers improved aptamer selection efficiencies through the application of highly stringent selection conditions: it retrieves a small number (<10(6)) of magnetic beads suspended in a large volume (>50 mL) and concentrates them into a microfluidic chamber (8 μL) with minimal loss for continuous washing. We performed three rounds of the VDC-MSELEX using streptavidin (SA) as the target and obtained new DNA aptamer sequences with low nanomolar affinity that specifically bind to the SA proteins.     

Thermo- and pH-sensitive IPN hydrogels based on PNIPAAm and PVA-Ma networks with LCST tailored close to human body temperature

Interpenetrating polymer network (IPN) hydrogels based on chemically modified poly(vinyl alcohol) (or PVA-Ma), with different degrees of substitution (DS), and poly(N-isopropylacrylamide) (or PNIPAAm) were obtained and characterized in this work. The PVA-Ma/PNIPAAm membrane hydrogels were prepared in two steps. In the first step the PVA-Ma hydrogels (with using PVA-Ma with different DS) were prepared by the reaction of double bonds on PVA-Ma, using the persulfate/TEMED pathway. In the second step the PNIPAAm network was prepared within the parent PVA-Ma network at different PVA-Ma/NIPAAm ratios using a photoreaction pathway. The studies show that degree of swelling of PVA-Ma/PNIPAAm IPN hydrogels is dependent on both temperature and pH of the soaking solution. The LCST of PVA-Ma/PNIPAAm IPN hydrogels, which was determined by measuring the intensity of light transmitted through the swollen hydrogels, can be tailored closer to human body temperature. Furthermore, SEM images showed that the IPN hydrogels present characteristic morphology as compared to parent PVA-Ma networks. IPN hydrogels presented lower cytotoxicity as compared to respective PVA-Ma hydrogels but the increase in the PVA-Ma/NIPAAm ratio allows the respective hydrogels being lesser cytocompatibles. The IPN hydrogels synthesized in this work presented characteristics that potentize their application as biomaterials, drug carriers, artificial muscles and treatment of wound.     

A Time‐Programmed Release of Dual Drugs from an Implantable Trilayer Structured Fiber Device for Synergistic Treatment of Breast Cancer

Combination chemotherapy with time‐programmed administration of multiple drugs is a promising method for cancer treatment. However, realizing time‐programmed release of combined drugs from a single carrier is still a great challenge in enhanced cancer therapy. Here, an implantable trilayer structured fiber device is developed to achieve time‐programmed release of combined drugs for synergistic treatment of breast cancer. The fiber device is prepared by a modified microfluidic‐electrospinning technique. The glycerol solution containing chemotherapy agent doxorubicin (Dox) forms the internal periodic cavities of the fiber, and poly(l ‐lactic acid) and poly(ε‐caprolactone) containing the angiogenesis inhibitor apatinib (Apa) form the double walls of the fiber. Rapid release of Dox can be obtained by adjusting the wall thickness of the cavities, meanwhile sustained release of Apa is achieved through the slow degradation of the fiber matrix. After the fiber device is implanted subcutaneously near to the implanted solid tumor of mice, an excellent synergistic therapeutic effect is achieved through time‐programmed release of the combined dual drugs. The fiber device provides a platform to sequentially co‐deliver dual or multiple drugs for enhanced combined therapeutic efficacy.     

Synthesis and characterization of partially biodegradable and thermosensitive hydrogel

A partially biodegradable and thermosensitive hybrid hydrogel network (DAN series) based on dextran-allylisocyanate (Dex-AI) and poly(N-isopropylacrylamide) (PNIPAAm) was synthesized via UV photocrosslinking. These hybrid hydrogels were characterized in terms of their chemical structure, thermal, mechanical, morphological and temperature-induced swelling properties. The effect of the composition ratio of Dex-AI to PNIPAAm on such properties were examined. The differential scanning calorimetry data show that this Dex-AI/PNIPAAm hybrid network has an increased lower critical solution temperature (LCST) and glass transition temperature (Tg) with an increase in the Dex-AI content. The interior morphology of these hybrid hydrogels revealed a decreased porous microstructure with an increase in the Dex-AI content in the hybrid network. Furthermore, if the Dex-AI composition became too high, a distinctive network structure with two different microporous structures appeared. The mechanical properties of these hybrid hydrogels also increased with an increase in the Dex-AI content. The temperature dependence of the swelling ratio, the deswelling kinetics as well as the reswelling kinetics was also characterized by gravimetric method. When comparing with a normal PNIPAAm hydrogel, these Dex-AI/PNIPAAm hybrid networks, due to the presence of Dex-AI moiety, also show improved temperature-induced intelligent properties, such as the faster and controllable response dynamics, which may find promising applications in a wide variety of fields, such as biomedical and bioengineering fields.     

Release of BSA from porous matrices constituted of alginate–Ca and PNIPAAm-interpenetrated networks

The synthesis of thermosensitive Interpenetrating Polymer Network (IPN) hydrogels and the release of Bovine Serum Albumin (BSA) from the hydrogels were reported. The hydrogels, constituted of poly(N-isopropyl acrylamide) PNIPAAm network interpenetrated in alginate–Ca²⁺ network, were synthesized in a two-stepped process. In the first step, PNIPAAm network was synthesized from an aqueous solution containing N-isopropyl acrylamide (NIPAAm) monomers and N,N′-methylene-bis-acrylamide (MBAAm) co-monomers, and sodium alginate (SA) (1 or 2% w/v). The concentration of NIPAAm monomers in the hydrogel-forming solution was always 2.5, 5.0 or 10.0% (w/v). In the second step, alginate–Ca²⁺ networks were formed by immersion of the membrane, obtained on the first step, in a 1.0% (w/v) aqueous calcium chloride. The IPN hydrogels were characterized as a function of temperature (from 25 to 45 °C) through the following measurements: drop water contact angle (DWCA), compression elastic modulus (E) and cross-linking density (ν_e). The morphology was investigated using scanning electronic microscopy (SEM). In vitro release of BSA from the hydrogels was monitored by UV–Vis spectroscopy at 22 °C and 37 °C. DWCA results showed a decrease in the hydrogel hydrophilicity when the temperature and/or the PNIPAAm amount on hydrogels were increased. PNIPAAm-loader hydrogels are more compacted and presented elevated rigidity, mainly above 35 °C. This trend was attributed to the collapsing of PNIPAAm chains as the hydrogels were warmed above its Lower Critical Solution Temperature (LCST), which in aqueous solution is ca. 32–33 °C. The amount of BSA released from the alginate–Ca²⁺/PNIPAAm hydrogels changes inversely to both amount of PNIPAAm and temperature. The transport of BSA from the hydrogels was evaluated through a conventional model. In the lesser-compacted hydrogels the release occurs mostly by diffusion. In the more compacted ones the chain relaxation contributes to the BSA release. Thus, the alginate–Ca²⁺/PNIPAAm IPN-typed matrixes may be considered as smart hydrogels for the release of BSA, because the amount and rate of BSA released may be tailored by both the NIPAAm concentration in the hydrogel-forming solution and the control of temperature of hydrogel.     

pH- and temperature-sensitive polymeric microspheres for drug delivery: the dissolution of copolymers modulates drug release

Most pH-/temperature-responsive polymers for controlled release of drugs are used as cross-linked hydrogels. However, the solubility properties of the linear polymers below and above the lower critical solution temperature (LCST) are not exploited. Here, the preparation and characterization of poly (N-isopropylacrylamide-co-methacrylic acid-co-methyl methacrylate) (poly (NIPAAm-co-MA-co-MM)) and poly (N-isopropylacrylamide-co-acrylamide) (poly (NIPAAm-co-AAm)), known as “smart” polymers (SP), is reported. Both poly (NIPAAm-co-MA-co-MM) and poly (NIPAAm-co-AAm) display pH- and temperature-responsive properties. Poly (NIPAAm-co-MA-co-MM) was designed to be insoluble in the gastric fluid (pH = 1.2), but soluble in the intestinal fluid (pH = 6.8 and 7.4), at the body temperature (37°C). Poly (NIPAAm-co-AAm) was designed to have a lower critical solution temperature (LCST) corresponding to 37°C at pH = 7.4, therefore it is not soluble above the LCST. The solubility characteristics of these copolymers were exploited to modulate the rate of release of drugs by changing pH and/or temperature. These copolymers were solubilized with hydrophobic cellulose acetate butyrate (CAB) and vitamin B₁₂ (taken as a water soluble drug model system) in an acetone/methanol mixture and dispersed in mineral oil. By a progressive evaporation of the solvent, the liquid droplets were transformed into loaded CAB/SP microspheres. Differential scanning calorimetric studies and scanning electron microscopy analysis demonstrated that the polymeric components of the microspheres precipitated separately during solvent evaporation forming small microdomains. Moreover, vitamin B₁₂ was found to be molecularly dispersed in both microdomains with no specific affinity for any polymeric component of microspheres. The release of vitamin B₁₂ was investigated as a function of temperature, pH, and the CAB/SP ratio.     

Properties and drug release profile of poly(N-isopropylacrylamide) microgels functionalized with maleic anhydride and alginate

This study highlights the advantages of functionalized poly(N-isopropylacrylamide) (PNIPAAm) microgels over pure PNIPAAm microgels in terms of polymer network properties and drug release profiles. PNIPAAm network was modified by addition of maleic anhydride (MA) as a comonomer and by formation of interpenetrating polymer network in the presence of alginate. The functionalized thermosensitive microgels in the size range from 20 to 80 μm and with better performance in comparison with pure PNIPAAm microgels were prepared by inverse suspension polymerization. The impact of MA and alginate on the PNIPAAm microgel structure was evaluated through analysis of microgel size, size distribution, volume phase transition temperature (VPTT), equilibrium swelling ratio as well as morphology of the system. It was shown that the controlled modification of PNIPAAm network could result in microgels of considerably improved swelling capacity with unchanged thermosensitivity and maintained open pore morphology. In addition, drug release behavior of microgels could be markedly altered. Release of procaine hydrochloride from the selected microgels was studied using Franz diffusion cell at temperatures below and above VPTT of the microgels. Temperature-controlled drug release pattern was dependent on the type of functionalization of PNIPAAm network. According to drug loading properties and drug release mechanism, PNIPAAm/MA copolymer microgels demonstrated the optimal performances.     

Sequential determination of two proteins by temperature-triggered homogeneous chemiluminescent immunoassay

A novel protocol for performing a sequential dual-protein immunoassay, based on a temperature-triggered separation/mixing process and HRP-catalyzed chemiluminescence (CL) detection, is described. In contrast to current multilabel-based detection techniques, a single HRP label is employed in this proposed method. Herein we introduce poly(N-isopropylacrylamide) (PNIP) and magnetic beads as bimolecular immobilizing carriers to separate different targets by taking advantage of thermal response, as demonstrated by sequential detection of human IgG and IgA. PNIP is known to aggregate and precipitate out of water when the temperature is raised above the lower critical solution temperature (LCST) of 31 degrees C; thus, it can be separated from supernatant by centrifugation. Besides, magnetic beads can be separated from PNIP by magnetic force as the temperature is lower than LCST. A homogeneous noncompetitive ELISA was employed, formed by primary antibodies immobilized onto the surface of magnetic beads and PNIP, antigen as IgG and IgA in the sample, and HRP-labeled second antibodies. Moreover, highly sensitive CL detection of HRP was applied, and the detection limits of IgG and IgA were as low as 2.0 and 1.5 ng/mL, respectively. Within the calibrated amount, the protocol had excellent precision within 11% for each target and was comparable in performance to commercial single-analyte ELISAs. Furthermore, the proposed method has been successfully applied to the determination of dual analyte in real samples without cross-reaction, and a good correlation was achieved after comparison with the conventional assay for IgG and IgA in 40 human serum samples.