Selective Separation And Enrichment Of Hemoglobin Bovine From Blood By Metal-modified Carbon-based Solid Phase Extraction Adsorbent

Hemoglobin（Hb）is a protein rich in histidine and plays a vital role in many physiological activities.As an iron-containing transport protein,Hb transports oxygen and carbon dioxide in organisms.The content of Hb is related to the health of organisms,and its imbalance will lead to various diseases,such as anemia,leukemia,diabetes and heart disease.Therefore,accurate determination of Hb content is crucial for clinicopathological analysis.Because of the high similarity between Bovine hemoglobin（BHb）and Hb,it has become an ideal substitute for Hb.Therefore,it is necessary to develop an effective method for separating and enriching BHb in complex biological samples.In this work,two novel nanomaterials were prepared based on solid phase extraction technology,and the selective recognition and separation mechanism of the two materials for BHb was studied.The specific research contents include the following two parts:Firstly,a carbon-based Cu²⁺-immobilized metal-organic framework-modified molecularly imprinted polymer（C@GI@Cu-MOFs@MIPs）adsorbent was prepared using bovine hemoglobin（BHb）as a template molecule with carbon spheres as carriers for the selective separation and enrichment of BHb.This paper presents a systematic investigation of the influence of sample concentration,sample p H,ionic strength,and eluent type on the adsorption performance of the prepared adsorbent.It was experimentally proven that the adsorbent could be reused even after six adsorption-desorption cycles without significant deterioration in the activity.Moreover,the C@GI@Cu-MOFs@MIPs have good binding behavior toward BHb with fast binding kinetics（45 min）,high adsorption capacity(1051.1 mg g^-1),and satisfactory selectivity in the presence of interfering proteins.In addition,C@GI@Cu-MOFs@MIPs were successfully applied to selectively identify,separate,and enrich BHb from real bovine blood samples as confirmed by SDS-PAGE,and thus are expected to be used in the pretreatment of proteomic analysis.Secondly,an amino acid-immobilized copper ion-modified carbon-based adsorbent（C@TA@P@A-Cu）was prepared for selective bovine hemoglobin（BHb）adsorption in biological samples.Carbon nanoparticles were used as the matrix,and copper ions were attached to the amino acid-modified carbon nanoparticles as metal chelate complexes via immobilized metal affinity.BSA,Lyz,OVA,and HRP were chosen as reference proteins for further study.Furthermore,the synthesis conditions of adsorbents,SPE conditions,selectivity,competitivity,reproducibility,and reusability were extensively investigated.The results showed that the maximum adsorption capacity of C@TA@P@A-Cu microspheres for BHb under optimal conditions was673.0 mg g^–1.The addition of a Ti O₂ layer with an increased specific surface area of the adsorbent and the addition of poly-l-lysine（PLL）inhibited the adsorbent’s binding ability to non-BHb proteins,but chelated Cu²⁺increased the adsorbent’s specific binding ability to BHb.Furthermore,after six adsorptiondesorption cycles,the adsorbent has satisfactory reusability with no significant change in adsorption capacity.Furthermore,C@TA@P@A-Cu was successfully used to identify BHb from real blood samples,as confirmed by SDS-PAGE,and it is expected to have potential applications in protein purification and disease diagnosis.